Inhibitor of apoptosis proteins and nucleic acids and methods for making and using them

ABSTRACT

The invention provides polypeptides comprising inhibitor of apoptosis protein (IAP) family members, such as BmIAP initially derived from  Bombyx mori  BmN cells, and nucleic acids encoding them, and methods for making and using these compositions, including their use for inhibiting apoptosis.

PRIORITY INFORMATION

[0001] This application claims priority to U.S. Provisional ApplicationSerial No. 60/260,478, filed Jan. 8, 2001.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

[0002] This invention was made in part with Government support underNational Institutes of Health grants ES02701, AG15402, ES 04699, CA30199(NCI); U.S. Department of Agriculture grants 97-35302-4406, 9802852;Binational Agriculture Research and Development grant 96-34339-3532;and, National Institute of Environmental Health Science grant ES 05707.The Government may have certain rights in the invention.

TECHNICAL FIELD

[0003] This invention generally pertains to the fields of cell biologyand molecular biology. In particular, this invention providespolypeptides comprising the inhibitor of apoptosis protein (IAP) familymember BmIAP, initially derived from silkworm Bombyx mori BmN cells, andnucleic acids encoding them, and methods for making and using thesecompositions, including their use for inhibiting caspase proteases andapoptosis.

BACKGROUND

[0004] Apoptosis or programmed cell death is a cellular suicide processin which damaged or harmful cells are eliminated from multicellularorganisms. Cells undergoing apoptosis have distinct morphologicalchanges including cell shrinkage, membrane blebbing, chromatincondensation, apoptotic body formation and fragmentation. This cellsuicide program is evolutionarily conserved across animal and plantspecies. Apoptosis plays an important role in the development andhomeostasis of metazoans and is also critical in insect embryonicdevelopment and metamorphosis. Furthermore, apoptosis acts as a hostdefense mechanism. For example, virally infected cells are eliminated byapoptosis to limit the propagation of viruses. Apoptosis mechanisms areinvolved in plant reactions to biotic and abiotic insults. Dysregulationof apoptosis has been associated with a variety of human diseasesincluding cancer, neurodegenerative disorders and autoimmune diseases.Accordingly, identification of novel mechanisms to manipulate apoptosisprovides new means to study and manipulate this process.

[0005] The first “inhibitor of apoptosis protein” (IAPs) was identifiedin a baculovirus. The baculovirus LAPs, CpIAP and OpIAP, are able toblock apoptosis induced by p35-deficient baculovirus AcMNPV in insectSf-21 cells. Cellular IAP homologues have been found in various animalspecies including worms, insects and humans. IAP proteins have adistinctive primary structure. They contain one to three copiesof“baculoviral LAP repeat” (BIR) domain and most IAPs also contain aRING domain near their C-termini. The BIR domain contains a highlyconserved arrangement of Cys/His residues forming a stable fold thatchelates zinc. The BIR region was also found to interact with regulatorsof IAPs including Grim, Reaper and Hid from Drosophila and may alsomediate homo-oligomerization. The RING finger is a common zinc-bindingmotif that also exists in other cellular proteins. Recent studies showthat several IAPs, including XIAP (a NF-kappaB-dependent member of theLAP gene family, see, e.g., Deveraux (1999) EMBO J. 18:5242-5251),cIAP1, CIAP2, DIAP1, SfIAP and CpIAP are inhibitors of caspases, afamily of intracellular proteases responsible for the execution of theapoptosis program. These IAPs can directly bind and inhibit some membersof caspase family including caspases-3, -7 and -9. Structure-functionstudies have demonstrated that inhibition of caspases-3 and -7 requiresonly a single BIR domain while RING domain may perform other functionsincluding recruitment of ubiquitin conjugating enzymes (UBCs). Whenexpressed without the associated BIRs, the RING region of SfIAP wasfound to enhance the proapoptotic activity of mammalian caspase-9suggesting this domain operates as a trans-dominant inhibitor ofendogenous proteins involved in apoptosis suppression. Ectopicexpression of lepidopteran SfIAP and baculoviral CpIAP blocks apoptosisin mammalian cells, suggesting conservation of the apoptosis programamong various species and a shared mechanism used by the IAP family.

[0006]Bombyx mori (silkworm) has been domesticated for silk-productionfor thousands of years. Used together with baculoviruses, it has alsobeen developed as an organism for large-scale production of foreignproteins in the biotechnology industry. Despite its extensive use insericulture and biotechnology, to date no apoptosis-regulating genes ofsilkworm have been identified.

SUMMARY

[0007] The invention provides an isolated or recombinant nucleic acidcomprising a nucleic acid sequence having at least about 95% sequenceidentity, about 97% sequence identity, about 99% sequence identity toSEQ ID NO:1. In one aspect of the invention, the nucleic acid encodes apolypeptide capable of inhibiting apoptosis in insect cells, encodes apolypeptide capable of inhibiting apoptosis in insect cells, such aslepidopteran and coleopteran cells, e.g., Bombyx mori or Spodopterafrugiperda cells. In one aspect the nucleic acid encodes a polypeptidecapable of inhibiting apoptosis in mammalian cells, encodes apolypeptide capable of inhibiting apoptosis in plant cells, or encodes apolypeptide capable of inhibiting caspase 9, e.g., human caspase 9. Inothers aspects, the isolated or recombinant nucleic acid encodes apolypeptide having a sequence as set forth in SEQ ID NO:2, and,comprises a nucleic acid sequence as set forth in SEQ ID NO:1.

[0008] The invention provides an expression cassette (e.g., vector,recombinant virus) comprising at least one nucleic acid of the inventionoperably linked to a promoter. The nucleic acid can comprise a sequencehaving at least 95% sequence identity to SEQ ID NO:1. As defined herein,in one aspect, an expression cassette comprises a nucleic acid of theinvention operably linked to a promoter. The promoter can be aconstitutive or an inducible promoter, or, the promoter can be adevelopmentally regulated or a tissue specific promoter. In one aspect,the nucleic acid on the expression cassette encodes a polypeptide havinga sequence as set forth in SEQ ID NO:2.

[0009] The invention provides a transformed cell comprising a nucleicacid of the invention. This nucleic acid can comprise a sequence havingat least 95% sequence identity to SEQ ID NO:1. The cell can be amammalian cell (such as a human cell), an insect cell, such as aSpodoptera frugiperda or Bombyx mori cell, a plant cell, a bacteria, ayeast cell, and the like. The transformed cell can comprise a nucleicacid encoding a polypeptide having a sequence as set forth in SEQ IDNO:2. These transformed cells can be used in the screening methods ofthe invention, which provide for identification of modulators of thepolypeptides of the invention, or, compositions that specifically bindto the polypeptides of the invention.

[0010] The invention provides a non-human transgenic animal comprising anucleic acid sequence of the invention, e.g., one having at least 95%sequence identity to SEQ ID NO:1. The nonhuman transgenic animal can bea rat or a mouse. The nonhuman transgenic animal can comprise a nucleicacid encoding a polypeptide having a sequence as set forth in SEQ IDNO:2. The nucleic acid can encode a polypeptide capable of inhibitingapoptosis.

[0011] The invention provides a transgenic plant comprising a nucleicacid sequence of the invention, e.g., one having at least 95% sequenceidentity to SEQ ID NO:1. The transgenic plant can comprise a nucleicacid encoding a polypeptide capable of inhibiting apoptosis. Thetransgenic plant, as a result of expression of the nucleic acid of theinvention, can become abiotic or biotic insult resistant. The bioticinsult can be induced by a plant pathogen, such as a virus, a fungus, abacteria or a nemotode. The abiotic insult can be induced by highmoisture, low moisture, salinity, nutrient deficiency, air pollution,high temperature, low temperature, soil toxicity, herbicides orinsecticides. The transgenic plant, upon expressing a nucleic acid ofthe invention or being exposed to a polypeptide of the invention, can bephenotypically altered, e.g., wherein at least a portion of the plantexhibits a decreased level of senescence. The invention provides a seedcapable of germinating into a plant having in its genome a heterologousnucleic acid sequence comprising a nucleic acid of the invention, e.g.,one having at least 95% sequence identity to SEQ ID NO:1. The seed cancomprise a nucleic acid encoding a polypeptide capable of inhibitingapoptosis in a plant cell.

[0012] The invention provides an isolated or recombinant polypeptidecomprising a sequence having at least 95% sequence identity to SEQ IDNO:2. The isolated or recombinant polypeptide of the invention can becapable of inhibiting apoptosis in cells, e.g., in insect cells, such aslepidopteran cells, e.g., Bombyx mori or Spodoptera frugiperda cells,and coleopteran cells, in mammalian cells, in yeast cells, in bacterialcells, in plant cells. In one aspect, the isolated or recombinantpolypeptide of the invention is capable of inhibiting caspase 9. Theinvention provides an isolated or recombinant polypeptide comprising asequence as set forth in SEQ ID NO:2.

[0013] The invention provides a fusion protein comprising a polypeptideof the invention, e.g., a sequence having at least 95% sequence identityto SEQ ID NO:2, and a second domain. The fusion protein's second domaincan comprise glutathione S-transferase (GST), and other domains, asdescribed below.

[0014] The invention provides an antibody or binding fragment thereof,wherein the antibody or fragment specifically binds to a polypeptide oran immunogenic fragment thereof, wherein the polypeptide comprises asequence having at least 95% sequence identity to SEQ ID NO:2. Theinvention provides an antibody or binding fragment thereof, wherein theantibody or fragment specifically binds to a protein having an aminoacid sequence as set forth in SEQ ID NO:2 or an immunogenic fragmentthereof.

[0015] The invention provides an array comprising a nucleic acidcomprising a nucleic acid of the invention, e.g., a sequence having atleast 95% sequence identity to SEQ ID NO:1, or, a fragment thereof.

[0016] The invention provides a method of detecting or isolating apolypeptide, wherein the polypeptide comprises a sequence having atleast 95% sequence identity to SEQ ID NO:2, comprising contacting abiological sample with an antibody as set forth in claim 38 or claim 39.The invention provides a method of making a recombinant polypeptidecomprising expressing a nucleic acid comprising a sequence having atleast 95% sequence identity to SEQ ID NO:1.

[0017] The invention provides a method for inhibiting apoptosis in acell comprising the following steps: (a) providing an isolated orrecombinant polypeptide comprising a sequence having at least 95%sequence identity to SEQ ID NO:2, wherein the polypeptide is capableinhibiting apoptosis in the cell, and, (a) contacting the polypeptidewith the cell in an amount sufficient to inhibit apoptosis in the cell.The invention provides a method for inhibiting apoptosis in a cellcomprising the following steps: (a) providing an isolated or recombinantnucleic acid comprising a sequence having at least 95% sequence identityto SEQ ID NO:1, wherein the nucleic acid encodes polypeptide capable ofinhibiting apoptosis in the cell, and, (b) contacting the nucleic acidwith the cell and expressing the nucleic acid to produce an amount ofpolypeptide sufficient to inhibit apoptosis in the cell. In alternativeaspects of these methods the cell can be an insect cell, e.g., alepidopteran cell, such as a Bombyx mori cell or a Spodoptera frugiperdacell, and a coleopteran cell, a mammalian cell, or a plant cell.

[0018] The invention provides a method for identifying an agent that canmodulate the activity of a polypeptide, wherein the polypeptidecomprises a sequence having at least 95% sequence identity to SEQ IDNO:2 and is capable inhibiting a caspase 9 protease, comprising: (a)providing an isolated or recombinant polypeptide comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:2 that is capableinhibiting a caspase 9 protease, and a test agent, (b) contacting thecaspase 9 protease and polypeptide in the presence and absence of thetest agent; and, (c) measuring the ability of the polypeptide to inhibitthe caspase 9 protease in the presence and absence of the test agent,wherein an increase or decrease in the ability of the polypeptide toinhibit the caspase 9 protease in the presence of the test agentidentifies the test agent as a modulator of the polypeptide's activity.

[0019] The invention provides a method for identifying an agent that canmodulate the activity of a polypeptide, wherein the polypeptidecomprises a sequence having at least 95% sequence identity to SEQ IDNO:2 and is capable inhibiting apoptosis in a cell, comprising: (a)contacting a cell expressing the polypeptide recombinantly in thepresence and absence of a test agent before, during or after inducingapoptosis in the cell; and, (b) measuring the amount or degree of thepolypeptide's activity in the cell in the presence and absence of thetest agent, wherein an increase or decrease in the amount or degree ofapoptosis in the cell in the presence of the test agent identifies thetest agent as a modulator of the polypeptide's activity. In alternativeaspects of these methods the cell can be an insect cell, e.g., alepidopteran cell, such as a Bombyx mori cell or a Spodoptera frugiperdacell, or a coleopteran cell, a mammalian cell, a yeast cell, a bacterialcell, a plant cell, and the like. The degree of the polypeptide'sactivity in the cell can be determined by measuring the amount or degreeof apoptosis in the cell; the amount or degree of caspase proteaseactivity in the cell; the amount or degree of DNA fragmentation in thecell; the amount or degree of cleavage of substrates of caspases in thecell; or by measuring the amount or degree of any surrogate marker ofapoptosis in the cell.

[0020] The invention provides a method of generating an abiotic orbiotic insult-resistant plant comprising the following steps: (a)providing an isolated or recombinant polypeptide comprising a sequencehaving at least 95% sequence identity to SEQ ID NO:2, wherein thepolypeptide is capable inhibiting apoptosis in a plant cell, and, (a)contacting the polypeptide with the plant in an amount sufficient toinhibit apoptosis in the plant, thereby generating a plant that isbiotic insult resistant. The invention provides a method for generatingan abiotic or biotic insult-resistant plant comprising the followingsteps: (a) providing an isolated or recombinant nucleic acid comprisinga sequence having at least 95% sequence identity to SEQ ID NO:1, whereinthe nucleic acid encodes polypeptide capable of inhibiting apoptosis ina plant cell, and, (b) contacting the nucleic acid with the plant andexpressing the nucleic acid to produce an amount of polypeptidesufficient to inhibit apoptosis in the plant. In alternative aspects ofthese methods, the biotic insult is induced by a plant pathogen, such asa virus, a fungus, a bacteria or a nemotode. In alternative aspects ofthese methods, the abiotic insult is induced by high moisture, lowmoisture, salinity, nutrient deficiency, air pollution, hightemperature, low temperature, soil toxicity, herbicides or insecticides.

[0021] The details of one or more embodiments of the invention are setforth in the accompanying drawings and the description below. Otherfeatures, objects, and advantages of the invention will be apparent fromthe description and drawings, and from the claims.

[0022] All publications, patents, patent applications, GenBank sequencesand ATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

DESCRIPTION OF DRAWINGS

[0023]FIG. 1A is a schematic showing the location of the BIR domains(BIR1, residues 74 to 140; BIR2, residues 182 to 249; of SEQ ID NO:2, asset forth in Example 1, below) and the RING domain (residues 298 to 314;of SEQ ID NO:2) of BmIAP. FIG. 1B (SEQ ID NO: 2) is the full lengthamino acid sequence of BmIAP (also set forth in Example 1, below, as SEQID NO:2). Sequence alignments of the BIR1 (FIG. 1C) (SEQ ID NOS: 8-13,respectively in order of appearance), BIR2 (FIG. 1D) (SEQ ID NOS: 14-19,respectively in order of appearance) and RING (FIG. 1E) (SEQ ID NOS20-25, respectively in order of appearance) domains of BmIAP with thecorresponding domains of other IAP family members are shown. Bold textindicates identical amino acid. See Example 1, below.

[0024]FIG. 2 is a representation of photographs of Sf-21 cells takenthree days post-transfection at 40×magnification; the cells wereco-transfected with p35-deficient AcMNPV viral DNA and (FIG. 2A) BmIAP,or (FIG. 2D) SfIAP, (the production of occlusion bodies is indicated byarrowheads); (FIG. 2B) BmIAP-BIR, (FIG. 2C) BmIAP-RING; and, (FIG. 2E)AcIAP; in 2B, 2C and 2E the apoptotic body formation (without occlusionbody formation) are indicated by arrowheads. FIG. 2F depicts controluninfected SF-21 cells. Full details in Example 1, below.

[0025]FIG. 3 schematically summarizes the results of experiments showingthat recombinantly expressed BmIAP protects mammalian cells againstBax-induced but not Fas-induced apoptosis. Expression plasmid encodingBax (FIG. 3A) or Fas (FIG. 3B) were co-transfected into HEK 293 cellswith various myc-tagged IAP expression plasmids. In FIG. 3C recombinantBmIAP was added to cytosolic extracts from HEK293 cells concurrentlywith the addition of cytochrome-c and dATP. Full details in Example 1,below.

[0026]FIG. 4 schematically summarizes the results of experiments showingthat recombinant BmIAP directly suppresses caspase-9 but not caspase-3or caspase-7. FIG. 4A: Recombinant active caspase-9 was incubated withAc-LEHD-AFC substrate in the presence or absence of variousconcentration of recombinant purified BmIAP or SfIAP. FIG. 4B:Recombinant caspase-3 was incubated with Ac-DEVD-AFC substrate in thepresence or absence of GST-XIAP, GST-BmIAP or 0.5 uM GST-SfIAP. FIG. 4C:Recombinant caspase-7 was incubated with Ac-DEVD-AFC substrate in thepresence or absence of GST-XIAP, GST-BmIAP or GST-SfIAP. Full details inExample 1, below.

[0027] Like reference symbols in the various drawings indicate likeelements.

DETAILED DESCRIPTION

[0028] The invention provides polypeptides that are “inhibitors ofapoptosis protein (IAP) family members,” including the exemplary BmIAP(SEQ ID NO:2), and nucleic acids encoding them (e.g., SEQ ID NO:1), andmethods for making and using these compositions. BmIAP was initiallyderived from lepidopteran Bombyx mori BmN cells.

[0029] The polypeptides and nucleic acids of the invention can be usedfor inhibiting caspases, including insect, plant and mammalian caspases,such as human caspase 9, and “programmed cell death,” or apoptosis. Thepolypeptides of the invention have some amino acid sequence similaritywith SfIAP (see, e.g., Huang (2000) Proc. Natl. Acad. Sci. USA97:1427-1432), TnIAP and baculoviral CpIAP. As discussed herein,structure-function analysis of BmIAP (SEQ ID NO:2) reveals identicaldomain requirements (e.g. BIR plus RING, see FIG. 1A) for suppression ofapoptosis in both insect and mammalian cells, and similar caspaseselectivity (e.g. caspase-9) compared to the previously characterizedlepidopteran and baculovirus IAPs. These results strongly support theidea that lepidopteran IAPs are evolutionary conserved in both sequenceand function. Accordingly, the compositions and methods of the inventionare used to manipulate apoptotic (“cell death”) mechanisms in a varietyof cell types, including insect, plant and mammalian, such as human,cells, and organisms.

[0030] Drosophila and mammalian IAPs have been shown to play importantroles in the development of these organisms (see, e.g., Hay (1995) Cell83:1253-1262; Holcik (2000) Proc. Natl. Acad. Sci. USA 97(5),2286-2290). By analogy, the exemplary BmIAP (SEQ ID NO:2) is also acritical player in silkworm development. Accordingly, the compositionsand methods of the invention can be used to manipulate apoptosis duringthe development of the silkworm. Lepidopteran and coleopteran cells,e.g., Bombyx mori cells and Spodoptera frugiperda cells, are commonlyused in conjunction with expression vectors (recombinant viruses) toexpress large quantities of exogenous polypeptides (see, e.g., Juntunen(1999) Biochem J. 344 Pt 2:297-303); the compositions and methods of theinvention can be used to manipulate these expression systems. Thus, thecompositions and methods of the invention will have a significant impacton sericulture and biotechnology industries, particularly those relatedto the silkworm.

[0031] Example 1, below, describes the isolation and characterization ofa novel group of “Inhibitor of Apoptosis Proteins (IAP),” designatedBmIAP. An exemplary polypeptide of this BmIAP group has a sequence asset forth in SEQ ID NO:2; an exemplary nucleic acid encodes it having asequence as set forth in SEQ ID NO:1. The exemplary IAP polypeptide andnucleic acid of the invention were initially derived from Bombyx moriBmN cells. BmIAP (SEQ ID NO:2) contains two baculoviral IAP repeat (BIR)domains followed by a RING domain (the BIR domain contains a highlyconserved arrangement of Cys/His residues forming a stable fold thatchelates zinc and the RING finger is a common zinc-binding motif thatalso exists in other cellular proteins), see FIG. 1A. BmIAP shares somecommon structural and functional properties with lepidopteran IAPs,SfIAP and TnIAP, and with two baculoviral IAPs, CpIAP and OpIAP,suggesting evolutionary conservation.

[0032] The polypeptides of the invention, “BmIAP,” block programmed celldeath (apoptosis) in Spodoptera frugiperda Sf-21 cells induced by p35(apoptosis inhibiting)-deficient Autographa californicanucleopolyhedrovirus (AcMNPV). BmIAP's anti-apoptotic function requiresboth the BIR domains and RING domain. In mammalian cells, BmIAP inhibitsBax-induced but not Fas-induced apoptosis. The data discussed belowdemonstrates that BmIAP can inhibit mammalian caspase-9 (an initiatorcaspase in the mitochondria/cytochrome-c pathway), and may be a specificinhibitor of caspase 9, but not the downstream effector proteases,caspase-3 and caspase-7. While the invention is not limited by anyparticular mechanism of action, these data support the role of thepolypeptides of the invention in suppressing apoptosis as involvinginhibition of an upstream initiator caspase (e.g., mammalian caspase-9)in the conserved mitochondria/cytochrome-c pathway. Inhibition of suchcaspases effectively also inhibit apoptosis.

[0033] Definitions

[0034] Unless defined otherwise, all technical and scientific terms usedherein have the meaning commonly understood by a person skilled in theart to which this invention belongs. As used herein, the following termshave the meanings ascribed to them unless specified otherwise.

[0035] The term “antibody” or “Ab” includes both intact antibodieshaving at least two heavy (H) chains and two light (L) chainsinter-connected by disulfide bonds and antigen binding fragmentsthereof, or equivalents thereof, either isolated from natural sources,recombinantly generated or partially or entirely synthetic. Examples ofantigen binding fragments include, e.g., Fab fragments, F(ab′)2fragments, Fd fragments, dAb fragments, isolated complementaritydetermining regions (CDR), single chain antibodies, chimeric antibodies,humanized antibodies, human antibodies made in non-human animals (e.g.,transgenic mice) or any form of antigen binding fragment.

[0036] The terms “array” or “microarray” or “DNA array” or “nucleic acidarray” or “biochip” as used herein is a plurality of target elements,each target element comprising a defined amount of one or more nucleicacid molecules, including the nucleic acids of the invention,immobilized a solid surface for hybridization to sample nucleic acids,as described in detail, below. The nucleic acids of the invention can beincorporated into any form of microarray, as described, e.g., in U.S.Pat. Nos. 6,045,996; 6,022,963; 6,013,440; 5,959,098; 5,856,174;5,770,456; 5,556,752; 5,143,854.

[0037] A “biotic insult”, as used herein, refers to plant challengecaused by viable or biologic agents (biotic agents), such as insects,fungi, bacteria, viruses, nematodes, viroids, mycoplasmas, and the like.

[0038] An “abiotic insult”, as used herein, refers to plant challenge bya non-viable or non-living agent (abiotic agent). Abiotic agents thatcan cause an abiotic insult include, for example, environmental factorssuch as low moisture (drought), high moisture (flooding), nutrientdeficiency, radiation levels, air pollution (ozone, acid rain, sulfurdioxide, etc.), temperature (hot and cold extremes), and soil toxicity,as well as herbicide damage, pesticide damage, or other agriculturalpractices (e.g., over-fertilization, improper use of chemical sprays,etc.).

[0039] The term “expression cassette” refers to any recombinantexpression system for the purpose of expressing a nucleic acid sequenceof the invention in vitro or in vivo, constitutively or inducibly, inany cell, including, in addition to insect and plant cells, prokaryotic,yeast, fungal or mammalian cells. The term includes linear or circularexpression systems. The term includes all vectors. The cassettes canremain episomal or integrate into the host cell genome. The expressioncassettes can have the ability to self-replicate or not, i.e., driveonly transient expression in a cell. The term includes recombinantexpression cassettes that contain only the minimum elements needed fortranscription of the recombinant nucleic acid.

[0040] The term “heterologous” when used with reference to a nucleicacid, indicates that the nucleic acid is in a cell or plant where it isnot normally found in nature; or, comprises two or more subsequenceswhich are not found in the same relationship to each other as normallyfound in nature, or is recombinantly engineered so that its level ofexpression, or physical relationship to other nucleic acids or othermolecules in a cell, or structure, is not normally found in nature. Forinstance, a heterologous nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged ina manner not found in nature; e.g., a promoter sequence operably linkedto a nucleic of the invention. As another example, the inventionprovides recombinant constructs (expression cassettes, vectors, viruses,and the like) comprising various combinations of promoters and sequencesof the invention.

[0041] As used herein, “isolated,” when referring to a molecule orcomposition, such as, e.g., a nucleic acid or polypeptide of theinvention, means that the molecule or composition is separated from atleast one other compound, such as a protein, DNA, RNA, or othercontaminants with which it is associated in vivo or in its naturallyoccurring state. Thus, a nucleic acid sequence is considered isolatedwhen it has been isolated from any other component with which it isnaturally associated. An isolated composition can, however, also besubstantially pure. An isolated composition can be in a homogeneousstate. It can be in a dry or an aqueous solution. Purity and homogeneitycan be determined, e.g., using analytical chemistry techniques such as,e.g., polyacrylamide gel electrophoresis (SDS-PAGE) or high performanceliquid chromatography (HPLC).

[0042] The term “nucleic acid” or “nucleic acid sequence” refers to adeoxy-ribonucleotide or ribonucleotide oligonucleotide, includingsingle- or double-stranded forms, and coding or non-coding (e.g.,“antisense”) forms. The term encompasses nucleic acids containing knownanalogues of natural nucleotides. The term also encompassesnucleic-acid-like structures with synthetic backbones. DNA backboneanalogues provided by the invention include phosphodiester,phosphorothioate, phosphorodithioate, methylphosphonate,phosphoramidate, alkyl phosphotriester, sulfamate, 3′-thioacetal,methylene(methylimino), 3′-N-carbamate, morpholino carbamate, andpeptide nucleic acids (PNAs); see Oligonucleotides and Analogues, aPractical Approach, edited by F. Eckstein, IRL Press at OxfordUniversity Press (1991); Antisense Strategies, Annals of the New YorkAcademy of Sciences, Volume 600, Eds. Baserga and Denhardt (NYAS 1992);Milligan (1993) J. Med. Chem. 36:1923-1937; Antisense Research andApplications (1993, CRC Press). PNAs contain non-ionic backbones, suchas N-(2-aminoethyl) glycine units. Phosphorothioate linkages aredescribed, e.g., by U.S. Pat. Nos. 6,031,092; 6,001,982; 5,684,148; seealso, WO 97/03211; WO 96/39154; Mata (1997) Toxicol. Appl. Pharmacol.144:189-197. Other synthetic backbones encompassed by the term includemethylphosphonate linkages or alternating methylphosphonate andphosphodiester linkages (see, e.g., U.S. Pat. No. 5,962,674;Strauss-Soukup (1997) Biochemistry 36:8692-8698), and benzylphosphonatelinkages (see, e.g., U.S. Pat. No. 5,532,226; Samstag (1996) AntisenseNucleic Acid Drug Dev 6:153-156). The term nucleic acid is usedinterchangeably with gene, DNA, RNA, cDNA, mRNA, oligonucleotide primer,probe and amplification product.

[0043] As used herein the terms “polypeptide,” “protein,” and “peptide”are used interchangeably and include compositions of the invention thatalso include “analogs,” or “conservative variants” and “mimetics” (e.g.,“peptidominetics”) with structures and activity that substantiallycorrespond to the polypeptides of the invention, including the exemplarysequence as set forth in SEQ ID NO:2. Thus, the terms “conservativevariant” or “analog” or “mimetic” also refer to a polypeptide or peptidewhich has a modified amino acid sequence, such that the change(s) do notsubstantially alter the polypeptide's (the conservative variant's)structure and/or activity (e.g., ability to inhibit caspase 9, toinhibit apoptosis), as defined herein. These include conservativelymodified variations of an amino acid sequence, i.e., amino acidsubstitutions, additions or deletions of those residues that are notcritical for protein activity, or substitution of amino acids withresidues having similar properties (e.g., acidic, basic, positively ornegatively charged, polar or non-polar, etc.) such that thesubstitutions of even critical amino acids does not substantially alterstructure and/or activity. Conservative substitution tables providingfunctionally similar amino acids are well known in the art. For example,one exemplary guideline to select conservative substitutions includes(original residue followed by exemplary substitution): ala/gly or ser;arg/lys; asn/gln or his; asp/glu; cys/ser; gln/asn; gly/asp; gly/ala orpro; his/asn or gln; ile/leu or val; leu/ile or val; lys/arg or gln orglu; met/leu or tyr or ile; phe/met or leu or tyr; ser/thr; thr/ser;trp/tyr; tyr/trp or phe; val/ile or leu. An alternative exemplaryguideline uses the following six groups, each containing amino acidsthat are conservative substitutions for one another: 1) Alanine (A),Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3)Asparagine (N), Olutamine (Q); 4) Arginine (R), Lysine (K); 5)Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); (see also, e.g.,Creighton (1984) Proteins, W. H. Freeman and Company; Schulz and Schimer(1979) Principles of Protein Structure, Springer-Verlag). One of skillin the art will appreciate that the above-identified substitutions arenot the only possible conservative substitutions. For example, for somepurposes, one may regard all charged amino acids as conservativesubstitutions for each other whether they are positive or negative. Inaddition, individual substitutions, deletions or additions that alter,add or delete a single amino acid or a small percentage of amino acidsin an encoded sequence can also be considered “conservatively modifiedvariations.”

[0044] The terms “mimetic” and “peptidomimetic” refer to a syntheticchemical compound that has substantially the same structural and/orfunctional characteristics of the polypeptides of the invention (e.g.,ability to inhibit apoptosis, antigenicity, etc.). The mimetic can beeither entirely composed of synthetic, non-natural analogues of aminoacids, or, is a chimeric molecule of partly natural peptide amino acidsand partly non-natural analogs of amino acids. The mimetic can alsoincorporate any amount of natural amino acid conservative substitutionsas long as such substitutions also do not substantially alter themimetics-structure and/or activity. As with polypeptides of theinvention which are conservative variants, routine experimentation willdetermine whether a mimetic is within the scope of the invention, i.e.,that its structure and/or function is not substantially altered.Polypeptide mimetic compositions can contain any combination ofnon-natural structural components, which are typically from threestructural groups: a) residue linkage groups other than the naturalamide bond (“peptide bond”) linkages; b) non-natural residues in placeof naturally occurring amino acid residues; or c) residues which inducesecondary structural mimicry, i.e., to induce or stabilize a secondarystructure, e.g., a beta turn, gamma turn, beta sheet, alpha helixconformation, and the like. A polypeptide can be characterized as amimetic when all or some of its residues are joined by chemical meansother than natural peptide bonds. Individual peptidomimetic residues canbe joined by peptide bonds, other chemical bonds or coupling means, suchas, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctionalmaleimides, N,N′-dicyclohexylcarbodiimide (DCC) orN,N′-diisopropylcarbodiimide (DIC). Linking groups that can be analternative to the traditional amide bond (“peptide bond”) linkagesinclude, e.g., ketomethylene (e.g., —C(═O)—CH₂—for —C(═O)—NH—),aminomethylene (CH₂—NH), ethylene, olefin (CH═CH), ether (CH₂—O),thioether (CH₂—S), tetrazole (CN₄—), thiazole, retroamide, thioamide, orester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of AminoAcids, Peptides and Proteins, Vol. 7, pp 267-357, “Peptide BackboneModifications,” Marcell Dekker, N.Y.). A polypeptide can also becharacterized as a mimetic by containing all or some non-naturalresidues in place of naturally occurring amino acid residues;non-natural residues are well described in the scientific and patentliterature.

[0045] As used herein, a “pathogen” refers to any agent that causes adisease or disease state in an animal or plant, including, but notlimited to viruses, fungi, bacterium, nematodes, and other relatedmicroorganisms.

[0046] The term “plant” includes whole plants, plant parts (e.g.,leaves, stems, flowers, roots, etc.), plant protoplasts, seeds and plantcells and progeny of same. The class of plants which can be used in themethod of the invention is generally as broad as the class of higherplants amenable to transformation techniques, including angiosperms(monocotyledonous and dicotyledonous plants), as well as gymnosperms. Itincludes plants of a variety of ploidy levels, including polyploid,diploid, haploid and hemizygous states. Plantlets are also includedwithin the meaning of “plant”. Suitable plants for use in the inventioninclude any plants amenable to transformation techniques, including bothmonocotyledonous and dicotyledonous plants. Examples of monocotyledonousplants include, but are not limited to, asparagus, field and sweet corn,barley, wheat, rice, sorghum, onion, pearl millet, rye and oat, andornamentals. Examples of dicotyledonous plants include, but are notlimited to, tomato, potato, arabidopsis, tobacco, cotton, rapeseed,field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, colecrops or Brassica (e.g., cabbage, broccoli, cauliflower, brusselsprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash,melons, cantaloupe, sunflowers and various ornamentals. The term “plantcell”, as used herein, refers to protoplasts, gamete producing cells,and cells that are capable of regenerating into whole plants.Accordingly, a seed comprising multiple plant cells capable ofregenerating into a whole plant is included in the definition of “plantcell”. As used herein, “plant tissue” includes differentiated andundifferentiated tissues of a plant, including but not limited to roots,stems, shoots, leaves, pollen, seeds, tumor tissue and various forms ofcells and culture such as single cells, protoplast, embryos, and callustissue.

[0047] As used herein, “recombinant” refers to a polynucleotidesynthesized or otherwise manipulated in vitro (e.g., “recombinantpolynucleotide”), to methods of using recombinant polynucleotides toproduce gene products in cells or other biological systems, or to apolypeptide (“recombinant protein”) encoded by a recombinantpolynucleotide.

[0048] As used herein, the term “promoter” includes all sequencescapable of driving transcription of a coding sequence in a cell,including an insect cell, a plant cell, a mammalian cell, and the like.Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription.

[0049] The term percent “sequence identity,” in the context of two ormore nucleic acids or polypeptide sequences refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of nucleotides (or amino acid residues) that are the same,when compared and aligned for maximum correspondence over a comparisonwindow, as measured using one of the following sequence comparisonalgorithms or by manual alignment and visual inspection. This definitionalso refers to the complement (antisense strand) of a sequence. Forexample, in alternative embodiments, nucleic acids within the scope ofthe invention include those with a nucleotide sequence identity that isat least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99% of the exemplary sequence set forth in SEQID NO:1. In alternative embodiments, polypeptides within the scope ofthe invention include those with an amino acid sequence identity that isat least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99% of the exemplary sequences set forth inSEQ ID NO:2. Two sequences with these levels of identity are“substantially identical” and within the scope of the invention. Thus,if a nucleic acid sequence has the requisite sequence identity to SEQ IDNO:1, or a subsequence thereof, it also is a polynucleotide sequencewithin the scope of the invention. If a polynucleotide sequence has therequisite sequence identity to SEQ ID NO:2, or a subsequence thereof, italso is a polypeptide within the scope of the invention. In one aspect,the percent identity exists over a region of the sequence that is atleast about 25 nucleotides or amino acid residues in length, or, over aregion that is at least about 50 to 100 nucleotides or amino acids inlength. Parameters (including, e.g., window sizes, gap penalties and thelike) to be used in calculating “percent sequence identities” betweentwo nucleic acids or polypeptides to identify and determine whether oneis within the scope of the invention are described in detail, below.

[0050] Polypeptides and Peptides

[0051] The invention provides an isolated or recombinant polypeptidecomprising a sequence having at least 95% sequence identity to SEQ IDNO:2. One exemplary polypeptide comprises the sequence as set forth inSEQ ID NO:2, and fragments (e.g., antigenic fragments) thereof (as notedabove, the term polypeptide includes peptides and peptidomimetics,etc.). Polypeptides and peptides of the invention can be isolated fromnatural sources, be synthetic, or be recombinantly generatedpolypeptides. Peptides and proteins can be recombinantly expressed invitro or in vivo. The peptides and polypeptides of the invention can bemade and isolated using any method known in the art.

[0052] Polypeptide and peptides of the invention can also besynthesized, whole or in part, using chemical methods well known in theart. See e.g., Caruthers (1980) Nucleic Acids Res. Symp. Ser. 215-223;Horn (1980) Nucleic Acids Res. Symp. Ser. 225-232; Banga, A. K.,Therapeutic Peptides and Proteins, Formulation, Processing and DeliverySystems (1995) Technomic Publishing Co., Lancaster, Pa. For example,peptide synthesis can be performed using various solid-phase techniques(see e.g., Roberge (1995) Science 269:202; Merrifield (1997) MethodsEnzymol. 289:3-13) and automated synthesis may be achieved, e.g., usingthe ABI 431A Peptide Synthesizer (Perkin Elmer). The skilled artisanwill recognize that individual synthetic residues and polypeptidesincorporating mimetics can be synthesized using a variety of proceduresand methodologies, which are well described in the scientific and patentliterature, e.g., Organic Syntheses Collective Volumes, Gilman, et al.(Eds) John Wiley & Sons, Inc., N.Y. Polypeptides incorporating mimeticscan also be made using solid phase synthetic procedures, as described,e.g., by Di Marchi, et al., U.S. Pat. No. 5,422,426. Peptides andpeptide mimetics of the invention can also be synthesized usingcombinatorial methodologies. Various techniques for generation ofpeptide and peptidomimetic libraries are well known, and include, e.g.,multipin, tea bag, and split-couple-mix techniques; see, e.g., al-Obeidi(1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol.1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996)Methods Enzymol. 267:220-234. Modified peptides of the invention can befurther produced by chemical modification methods, see, e.g., Belousov(1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol.Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896.

[0053] The invention provides a fusion protein comprising a polypeptideof the invention, e.g., a sequence having at least 95% sequence identityto SEQ ID NO:2, and a second domain. Thus, peptides and polypeptides ofthe invention are synthesized and expressed as chimeric or “fusion”proteins with one or more additional domains linked thereto for, e.g.,to more readily isolate or identify a recombinantly synthesized peptide,and the like. Detection and purification facilitating domains include,e.g., metal chelating peptides such as polyhistidine tracts andhistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp, Seattle Wash.). The inclusion of acleavable linker sequences such as Factor Xa or enterokinase(Invitrogen, San Diego Calif.) between the purification domain andGCA-associated peptide or polypeptide can be useful to facilitatepurification. For example, an expression vector can include anepitope-encoding nucleic acid sequence linked to six histidine residuesfollowed by a thioredoxin and an enterokinase cleavage site (see, e.g.,Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998) Protein Expr.Purif. 12:404-14). The histidine residues facilitate detection andpurification while the enterokinase cleavage site provides a means forpurifying the epitope from the remainder of the fusion protein.

[0054] Nucleic Acids, Expression Vectors and Transformed Cells

[0055] The invention provides an isolated or recombinant nucleic acidcomprising a nucleic acid sequence having at least 95% sequence identityto SEQ ID NO:1, and expression cassettes (e.g., vectors), cells andtransgenic animals comprising the nucleic acids of the invention. As thegenes and vectors of the invention can be made and expressed in vitro orin vivo, the invention provides for a variety of means of making andexpressing these genes and vectors. One of skill will recognize thatdesired phenotypes associated with altered gene activity can be obtainedby modulating the expression or activity of the genes and nucleic acids(e.g., promoters) within the expression cassettes (e.g., vectors) of theinvention. Any of the known methods described for increasing ordecreasing expression or activity can be used for this invention. Theinvention can be practiced in conjunction with any method or protocolknown in the art, which are well described in the scientific and patentliterature.

[0056] The nucleic acid sequences of the invention and other nucleicacids used to practice this invention, whether RNA, cDNA, genomic DNA,vectors, viruses or hybrids thereof, may be isolated from a variety ofsources, genetically engineered, amplified, and/or expressedrecombinantly. Any recombinant expression system can be used, including,in addition to insect and bacterial cells, e.g., mammalian, yeast orplant cell expression systems.

[0057] Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g.,Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) FreeRadic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896;Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68:109;Beaucage (1981) Tetra. Lett. 22:1859; U.S. Pat. No. 4,458,066.

[0058] Techniques for the manipulation of nucleic acids, such as, e.g.,generating mutations in sequences, subcloning, labeling probes,sequencing, hybridization and the like are well described in thescientific and patent literature, see, e.g., Sambrook, ed., MOLECULARCLONING: A LABORATORY MANUAL (2ND ED.), Vols. 1-3, Cold Spring HarborLaboratory, (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed.John Wiley & Sons, Inc., New York (1997); LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACIDPROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed.Elsevier, N.Y. (1993).

[0059] The invention provides nucleic acids of the invention “operablylinked” to a transcriptional regulatory sequence. “Operably linked”refers to a functional relationship between two or more nucleic acid(e.g., DNA) segments. Typically, it refers to the functionalrelationship of a transcriptional regulatory sequence to a transcribedsequence. For example, a promoter is operably linked to a codingsequence, such as a nucleic acid of the invention, if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance. For example, in one embodiment, a promoteris operably linked to a nucleic acid sequence of the invention, asexemplified by SEQ ID NO:1.

[0060] The invention further provides cis-acting transcriptionalregulatory sequences, which, in vivo, are operably linked to the codingsequence for the exemplary polypeptide of the invention, SEQ ID NO:2,including promoters, comprising the genomic sequences 5′ (upstream) of atranscriptional start site (see SEQ ID NO:1) and intronic sequences. Thepromoters of the invention contain cis-acting transcriptional regulatoryelements involved in message expression. These promoter sequences may bereadily obtained using routine molecular biological techniques. Forexample, additional genomic (and promoter) sequences may be obtained byscreening Bombyx mori genomic libraries using nucleic acids of theinvention. For example, genomic sequence can be readily identified by“chromosome walking” techniques, as described by, e.g., Hauser (1998)Plant J 16:117-125; Min (1998) Biotechniques 24:398-400. Other usefulmethods for further characterization of promoter sequences include thosegeneral methods described by, e.g., Pang (1997) Biotechniques22:1046-1048; Gobinda (1993) PCR Meth. Applic. 2:318; Triglia (1988)Nucleic Acids Res. 16:8186; Lagerstrom (1991) PCR Methods Applic. 1:111;Parker (1991) Nucleic Acids Res. 19:3055. As is apparent to one ofordinary skill in the art, these techniques can also be applied toidentify, characterize and isolate any genomic or cis-acting regulatorysequences corresponding to or associated with the nucleic acid andpolypeptide sequences of the invention.

[0061] The invention provides oligonucleotide primers that can amplifyall or any specific region within a nucleic acid sequence of theinvention, particularly, the exemplary SEQ ID NO:1. The nucleic acids ofthe invention can also be mutated, detected, generated or measuredquantitatively using amplification techniques. Using the nucleic acidsequences of the invention (e.g., as in the exemplary SEQ ID NO:1), theskilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also known in the art,and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y.); ligase chain reaction (LCR) (see, e.g., Barringer (1990)Gene 89:117); transcription amplification (see, e.g., Kwoh (1989) Proc.Natl. Acad. Sci. USA, 86:1173); and, self-sustained sequence replication(see, e.g., Guatelli (1990) Proc. Natl. Acad. Sci. USA, 87:1874); Q Betareplicase amplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491; Burg (1996) Mol. Cell. Probes 10:257-271) and other RNApolymerase mediated techniques (e.g., NASBA, Cangene, Mississauga,Ontario).

[0062] Expression vectors capable of expressing the nucleic acids andpolypeptides of the invention in animal cells, including insect andmammalian cells, are well known in the art. Vectors which may beemployed include recombinantly modified enveloped or non-enveloped DNAand RNA viruses, e.g., from baculoviridiae, parvoviridiae,picornoviridiae, herpesveridiae, poxviridae, adenoviridiae,picornnaviridiae or alphaviridae. Insect cell expression systemscommonly use recombinant variations of baculoviruses and othernucleopolyhedrovirus, e.g., Bombyx mori nucleopolyhedrovirus vectors(see, e.g., Choi (2000) Arch. Virol. 145:171-177). For example,Lepidopteran and Coleopteran cells are used to replicate baculovirusesto promote expression of foreign genes carried by baculoviruses, e.g.,Spodoptera frugiperda cells are infected with recombinant Autographacalifornica nuclear polyhedrosis viruses (AcNPV) carrying aheterologous, e.g., a human, coding sequence (see, e.g., Lee (2000) J.Virol. 74:11873-11880; Wu (2000) J. Biotechnol. 80:75-83). See, e.g.,U.S. Pat. No. 6,143,565, describing use of the polydnavirus of theparasitic wasp Glyptapanteles indiensis to stably integrate nucleic acidinto the genome of Lepidopteran and Coleopteran insect cell lines. Seealso, U.S. Pat. Nos. 6,130,074; 5,858,353; 5,004,687.

[0063] Mammalian expression vectors can be derived from adenoviral,adeno-associated viral or retroviral genomes. Retroviral vectors caninclude those based upon murine leukemia virus (see, e.g., U.S. Pat. No.6,132,731), gibbon ape leukemia virus (see, e.g., U.S. Pat. No.6,033,905), simian immuno-deficiency virus, human immuno-deficiencyvirus (see, e.g., U.S. Pat. No. 5,985,641), and combinations thereof.Describing adenovirus vectors, see, e.g., U.S. Pat. Nos. 6,140,087;6,136,594; 6,133,028; 6,120,764. See, e.g., Okada (1996) Gene Ther.3:957-964; Muzyczka (1994) J. Clin. Invst. 94:1351; U.S. Pat. Nos.6,156,303; 6,143,548 5,952,221, describing AAV vectors. See also6,004,799; 5,833,993.

[0064] Expression vectors capable of expressing proteins in plants arewell known in the art, and can include, e.g., vectors from Agrobacteriumspp., potato virus X (see, e.g., Angell (1997) EMBO J. 16:3675-3684),tobacco mosaic virus (see, e.g., Casper (1996) Gene 173:69-73), tomatobushy stunt virus (see, e.g, Hillman (1989) Virology 169:42-50), tobaccoetch virus (see, e.g., Dolja (1997) Virology 234:243-252), bean goldenmosaic virus (see, e.g., Morinaga (1993) Microbiol Immunol. 37:471-476),cauliflower mosaic virus (see, e.g., Cecchini (1997) Mol. Plant MicrobeInteract. 10:1094-1101), maize Ac/Ds transposable element (see, e.g.,Rubin (1997) Mol. Cell. Biol. 17:6294-6302; Kunze (1996) Curr. Top.Microbiol. Immunol. 204:161-194), and the maize suppressor-mutator (Spm)transposable element (see, e.g., Schlappi (1996) Plant Mol. Biol.32:717-725); and derivatives thereof.

[0065] The invention provides a transformed cell comprising a nucleicacid of the invention. The cells can be mammalian (such as human),insect (such as Spodoptera frugiperda, Spodoptera exigua, Spodopteralittoralis, Spodoptera litura, Pseudaletia separata, Trichoplusia ni,Plutella xylostella, Bombyx mori, Lymantria dispar, Heliothis virescens,Autographica californica and other insect, particularly lepidopteran andcoleopteran, cell lines), plant, bacterial, yeast, and the like.Techniques for transforming and culturing cells are well described inthe scientific and patent literature; see, e.g., Weiss (1995) MethodsMol. Biol. 39:79-95, describing insect cell culture in serum-free media;Tom (1995) Methods Mol. Biol. 39:203-224; Kulakosky (1998) Glycobiology8:741-745; Altmann (1999) Glycoconj. J. 16:109-123; Yanase (1998) ActaVirol. 42:293-298; U.S. Pat. Nos. 6,153,409; 6,143,565; 6,103,526.

[0066] Transgenic Non-human Animals

[0067] The invention also provides transgenic animals, including mammalsand insects. Insects stably expressing the nucleic acids of theinvention can be used for, e.g., experiments studies on apoptosis,screening for modulators of caspases and apoptosis, manipulation ofinsect life cycles, such as Bombyx mori and its use in silk production.The nucleic acids of the invention can be expressed in a variety ofinsect larvae, e.g., Bombyx mori (see, e.g., Maeda (1985) Nature 315:592-594), Trichoplusia ni, the cabbage looper larvae (Medin (1990) Proc.Nat. Acad. Sci. USA 87: 2760-2764) and Manduca sexta, the tobaccohomworm (U.S. Pat. No. 5,471,858). See, e.g., Keshan (2000) J. InsectPhysiol 46:1061-1068; U.S. Pat. No. 5,118,616.

[0068] The invention also provides transgenic non-human mammals, e.g.,goats, rats and mice, comprising the chimeric nucleic acids of theinvention. These animals can be used, e.g., as in vivo models to studyapoptosis, or, as models to screen for modulators of caspase enzymeactivity in vivo. Transgenic non-human animals can be designed andgenerated using any method known in the art; see, e.g., U.S. Pat. Nos.6,156,952; 6,118,044; 6,111,166; 6,107,541; 5,959,171; 5,922,854;5,892,070; 5,880,327; 5,891,698; 5,639,940; 5,573,933, describing makingand using transgenic mice, rats, rabbits, sheep, pigs and cows. Seealso, e.g., Pollock (1999) J. Immunol. Methods 231:147-157, describingthe production of recombinant proteins in the milk of transgenic dairyanimals; Baguisi (1999) Nat. Biotechnol. 17:456-461, demonstrating theproduction of transgenic goats.

[0069] Transgenic Plants

[0070] The invention also provides transgenic plants, including seeds,expressing the nucleic acids and polypeptide of the invention. Nucleicacids may be introduced into the genome of the desired plant host by avariety of conventional techniques. For example, the DNA construct maybe introduced directly into the genomic DNA of the plant cell usingtechniques such as electroporation and microinjection of plant cellprotoplasts, or the DNA constructs can be introduced directly to planttissue using ballistic methods, such as DNA particle bombardment.

[0071] Alternatively, transformed plant cells can be generated by fusionof the recipient cells with bacterial protoplasts containing DNA, use ofDEAE dextran, polyethylene glycol precipitation, as described, e.g., inPaszkowski (1984) EMBO J. 3:2717-2722. DNA construct can be introduceddirectly into the genomic DNA of the plant cell using electroporation,as described, e.g., in Fromm (1985) Proc. Natl. Acad. Sci. USA 82:5824,or by microinjection of plant cell protoplasts, as described, e.g.,Schnorf (1991) Transgenic Res. 1:23-30.

[0072] Nucleic acids can be introduced directly to plant tissue usingballistic methods, such as DNA particle bombardment. Microprojectilebombardment to deliver DNA into plant cells is an alternative means oftransformation for the numerous species considered recalcitrant toAgrobacterium- or protoplast-mediated transformation methods. Forexample, see, e.g., Christou (1997) Plant Mol. Biol. 35:197-203;Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein (1987) Nature327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69, discussing use ofparticle bombardment to introduce transgenes into wheat; and Adam (1997)supra, for use of particle bombardment to introduce YACs into plantcells. For example, Rinehart (1997) supra, used particle bombardment togenerate transgenic cotton plants. Apparatus for accelerating particlesis described U.S. Pat. No. 5,015,580; and, the commercially availableBioRad (Biolistics) PDS-2000 particle acceleration instrument; see also,John, U.S. Pat. No. 5,608,148; and Ellis, U.S. Pat. No. 5,681,730,describing particle-mediated transformation of gymnosperms.

[0073] DNA can also be introduced in to plant cells using recombinantviruses. Plant cells can be transformed using viral vectors, such as,e.g., tobacco mosaic virus derived vectors (Rouwendal (1997) Plant Mol.Biol. 33:989-999), see Porta (1996) “Use of viral replicons for theexpression of genes in plants,” Mol. Biotechnol. 5:209-221.

[0074] Alternatively, the DNA constructs may be combined with suitableT-DNA flanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobacteriumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. Agrobacterium tumefaciens mediated transformation techniques,including disarming and use of binary vectors, are well described in thescientific literature. See, e.g., Horsch (1984) Science 233:496-498;Fraley (1983) Proc. Natl. Acad. Sci. USA 80:4803 (1983); Gene Transferto Plants, Potrykus, ed. (Springer-Verlag, Berlin 1995).

[0075] Alignment Analysis of Sequences

[0076] The nucleic acid sequences of the invention include genes andgene products identified and characterized by analysis using theexemplary nucleic acid and protein sequences of the invention, includingSEQ ID NO:1 and SEQ ID NO:2. For sequence comparison, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used unless alternativeparameters are designated herein. The sequence comparison algorithm thencalculates the percent sequence identity for the test sequence(s)relative to the reference sequence, based on the designated or defaultprogram parameters. A “comparison window”, as used herein, includesreference to a segment of any one of the number of contiguous positionsselected from the group consisting of from 25 to 600, usually about 50to about 200, more usually about 100 to about 150 in which a sequencemay be compared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned.

[0077] Methods of alignment of sequences for comparison are well-knownin the art. Optimal alignment of sequences for comparison can beconducted, e.g., by the local homology algorithm of Smith & Waterman,Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm ofNeedleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search forsimilarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444 (1988), by computerized implementations of these algorithms(CLUSTAL, GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection.

[0078] In one aspect, a CLUSTAL algorithm, such as the CLUSTAL Wprogram, is used to determine if a nucleic acid or polypeptide sequenceis within the scope of the invention; see, e.g., Thompson (1994) Nuc.Acids Res. 22:4673-4680; Higgins (1996) Methods Enzymol 266:383-402.Variations can also be used, such as CLUSTAL X, see Jeanmougin (1998)Trends Biochem Sci 23:403-405; Thompson (1997) Nucleic Acids Res25:4876-4882. CLUSTAL W program, described by Thompson (1994) supra, inthe methods of the invention used with the following parameters: K tuple(word) size: 1, window size: 5, scoring method: percentage, number oftop diagonals: 5, gap penalty: 3.

[0079] Another algorithm is PILEUP, which can be used to determinewhether a polypeptide or nucleic acid has sufficient sequence identityto SEQ ID NO:1 or SEQ ID NO:2 to be with the scope of the invention.This program creates a multiple sequence alignment from a group ofrelated sequences using progressive, pairwise alignments to showrelationship and percent sequence identity. It also plots a tree ordendogram showing the clustering relationships used to create thealignment. PILEUP uses a simplification of the progressive alignmentmethod of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987). The methodused is similar to the method described by Higgins & Sharp, CABIOS5:151-153 (1989). The following parameters are used with PILEUP in themethods of the invention: default gap weight (3.00), default gap lengthweight (0.10), and weighted end gaps.

[0080] Another example of an algorithm that is suitable for determiningpercent sequence identity (i.e., substantial similarity or identity) inthis invention is the BLAST algorithm, which is described in Altschul(1990) J. Mol. Biol. 215:403-410. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold (Altschul (1990) supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits are then extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues,always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. In one embodiment, to determineif a nucleic acid sequence is within the scope of the invention, theBLASTN program (for nucleotide sequences) is used incorporating asdefaults a wordlength (W) of 11, an expectation (E) of 10, M=5, N=4, anda comparison of both strands. For amino acid sequences, the BLASTPprogram uses as default parameters a wordlength (W) of 3, an expectation(E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915).

[0081] Antibodies

[0082] The invention provides antibodies that specifically bind to thepolypeptides of the invention, e.g., the exemplary SEQ ID NO:2. Theseantibodies can be used, e.g., to isolate the polypeptides of theinvention, to identify the presence of polypeptides that are associatedwith apoptosis, and the like. To generate antibodies, polypeptides orpeptides (antigenic fragments of SEQ ID NO:2) can be conjugated toanother molecule or can be administered with an adjuvant. The codingsequence can be part of an expression cassette or vector capable ofexpressing the imimunogen in vivo (see, e.g., Katsumi (1994) Hum. GeneTher. 5:1335-9). Methods of producing polyclonal and monoclonalantibodies are known to those of skill in the art and described in thescientific and patent literature, see, e.g., Coligan, CURRENT PROTOCOLSIN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASIC ANDCLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif.; Goding, MONOCLONAL ANTIBODIES: PRINCIPLES AND PRACTICE (2d ed.)Academic Press, New York, N.Y. (1986); Harlow (1988) ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publications, New York.

[0083] Antibodies also can be generated in vitro, e.g., usingrecombinant antibody binding site expressing phage display libraries, inaddition to the traditional in vivo methods using animals. See, e.g.,Huse (1989) Science 246:1275; Ward (1989) Nature 341:544; Hoogenboom(1997) Trends Biotechnol. 15:62-70; Katz (1997) Annu. Rev. Biophys.Biomol. Struct. 26:27-45. Human antibodies can be generated in miceengineered to produce only human antibodies, as described by, e.g., U.S.Pat. Nos. 5,877,397; 5,874,299; 5,789,650; and 5,939,598. B-cells fromthese mice can be immortalized using standard techniques (e.g., byfusing with an immortalizing cell line such as a myeloma or bymanipulating such B-cells by other techniques to perpetuate a cell line)to produce a monoclonal human antibody-producing cell. See, e.g., U.S.Pat. Nos. 5,916,771; 5,985,615.

[0084] Measuring Markers of Apoptosis in a Cell

[0085] The invention provides a method for identifying an agent that canmodulate the activity of a polypeptide of the invention (e.g., apolypeptide having a sequence at least 95% sequence identity to SEQ IDNO:2) that is capable inhibiting apoptosis in a cell. The methodcomprises contacting a cell expressing the polypeptide recombinantly inthe presence and absence of a test agent before, during or afterinducing apoptosis in the cell; and, measuring the amount or degree ofthe polypeptide's activity in the cell in the presence and absence ofthe test agent, wherein an increase or decrease in the amount or degreeof apoptosis in the cell in the presence of the test agent identifiesthe test agent as a modulator of the polypeptide's activity.

[0086] The degree of the polypeptide's activity in the cell can bedetermined by measuring the amount or degree of apoptosis in the cell;the amount or degree of caspase protease activity in the cell; theamount or degree of DNA fragmentation in the cell; the amount or degreeof cleavage of substrates of caspases in the cell; or by measuring theamount or degree of any surrogate marker of apoptosis in the cell;methods for making these measurements are well known in the art, and theinvention incorporates all such methods and variations thereof. Forexample, see Methods in Enzymology (2000) volume 322, edited by John C.Reed, Academic Press, e.g., chapters on pages 3, 15, 41, describingassays to measure apoptosis and surrogate markers of apoptosis andenzymes with activity related to levels of apoptosis, e.g., assays todetermine DNA fragmentation, caspase assays, measuring annexin V (see,e.g., Zhang (1997) Biotechniques 23:525-531), and the like. See, e.g.,van Engeland (1996) Cytometry 24:131-139; Gorczyca (1998) Methods Mol.Biol. 91:217-238. See also, e.g., U.S. Pat. Nos. 6,165,737; 6,165,732;6,160,095; 6,143,522; 6,087,384; 6,077,684; 6,060,238; 6,054,436;5,985,829; 5,976,786; 5,952,189.

EXAMPLES

[0087] The following example is offered to illustrate, but not to limitthe claimed invention.

Example 1 Cloning, Recombinant Expression and Characterization of BmIAP

[0088] The following example describes the initial cloning of the BmIAPof the invention, recombinant expression of BmIAP nucleic acids andpolypeptides and characterization of the BmIAP of the invention.

[0089] Cloning of BmIAP: mRNA was isolated from BmN cells by using a kitfrom Qiagen, Inc. (Valencia, Calif.). Degenerate primers were used; theywere designed according to the consensus amino acid sequences betweenbaculoviral IAPs and Drosophila IAPs, having the sequence5′-GC(A/C/G/T)GA(A/C/G/T)GC(A/C/G/T)GG(A/C/G/T) TT(T/C)TT(T/C)TA-3′ (SEQID NO:3) and 5′-AC(A/C/G/T)AC(A/G)TG(A/C/G/T)CC (A/G)CA(A/C/G/T)GG-3′(SEQ ID NO:4). Reverse transcription-PCR was performed for 35 cycles byusing 94° C. for 45 sec, 46° C. for 1 min and 72° C. for 1 min.Amplified were blunt-end-cloned into HincII site of pTZ-19 and thensequenced. To obtain full-length BmIAP cDNAs, 5′ RACE and 3′ RACE wereperformed by using commercial kits (GIBCO/BRL; Takara) and5′-CTGTTCCCACGGAACGTC-3′ (SEQ ID NO:5) and 5′-GCCACCAATGATTCGAC-3′ (SEQID NO:6) as internal PCR primers.

[0090] Plasmid construction: A cDNA fragment encompassing the completeORF of BmIAP was PCR-amplified and subcloned into the EcoRI-XhoI sitesin pcDNA3-myc and pGEX4T-1. Plasmids encoding fragments of BmIAP,including BIR1+2 (amino acid residue 1-292) (SEQ ID NO:2) and RING(residue 293-346) (SEQ ID NO:2), were amplified by PCR by using primerscontaining either start or stop codons as appropriate and subcloned intopTZ-19 and pcDNA3-myc plasmids.

[0091] Protein expression and purification: pGEX4T-1-BmIAP plasmid wasintroduced into E. coli strain BL21 (DE3) containing the plasmid pT-Trx.Glutathione S-transferase (GST) fusion proteins were obtained byinduction with 0.05 mM isopropyl β-thiogalactoside at 25° C. for 8 hrand then purified using glutathione-Sepharose (see, e.g., Huang (2000)Proc. Natl. Acad. Sci. USA 97:1427-1432). The catalytic domains ofcaspase-3, caspase-7 and caspase-9 were expressed, purified byNi-chelation affinity-chromatography, and quantified as described byStennicke (1997) J. Biol. Chem. 272:25719-25723; Stennicke (1998)J.Biol. Chem 273:27084-27090; Stennicke (19999) J. Biol. Chem.274:8359-8362.

[0092] Cell extracts and Caspase assays: Cytosolic extracts wereprepared by using human embryonic kidney (HEK) 293 cells (see, e.g.,Deveraux (1997) Nature 388:300-303). For initiating caspase activation,1 uM horse heart cytochrome-c (Sigma) and 1 mM dATP was added toextracts, as described by Deveraux (1998) Embo J. 17:2215-2223). Caspaseactivity was assayed by release of 7-amino-4-trifluoromethyl-coumarin(AFC) from Ac-DEVD-AFC or Ac-LEHD-AFC (Calbiochem), using aspectrofluorimeter as described by Stennicke (1997) supra; Quan (1995)J. Biol. Chem. 270:10377-10379.

[0093] Cell culture, Transfection and Apoptosis Assays: Insect Sf-21cells were maintained at 27° C. in Excell 401 medium (J R H Biosciences,Lenexa, Kans.) supplemented with 2.5% FBS. vP35del, Autographacalifornica nuclear polyhedrosis virus (AcMNPV) containing a deletion inthe apoptosis suppressor “p35” gene (35-kilodalton protein gene) (see,e.g., Lerch (1993) Nucleic Acids Res. 21:1753-1760) was propagated inTN-386 cells (see, e.g., Clem (1991) Science 254:1388-1390). Plasmidsencoding full-length or deletion mutants of BmIAP (1 ug) wereco-transfected with 1 ug vP35del viral DNA into Sf-21 cells by usingLipofectin from GIBCO/BRL. Occlusion body formation was observed underlight-microscopy 3 days post-transfection. HEK293 cells were maintainedin DMEM (Irvine Scientific) supplemented with 10% FBS, 1 mM L-glutamine,and antibiotics. 293 cells (10⁶) were cotransfected by using Superfect™(Qiagen) with 0.1 ug of green fluorescence protein (GFP) marker plasmidpEGFP (CLONTECH), 0.25 ug of either pcDNA3-Bax or pcDNA3-Fas and 1.5 ugof pcDNA3-myc-BmIAP. Both floating and adherent cells were recovered 24to 36 hour post-transfection and pooled, and the percentage ofGFP-positive cells with nuclear apoptotic morphology was determined bystaining with 0.1 ug/ml 4′-6-diamidino-2-phenylindole (DAPI) (mean +/−S.D.; n=3) as described by Takhashi (1998) J. Biol. Chem. 273:7787-7790.

[0094] The full-length BmIAP cDNA (SEQ ID NO:1) and the BmIAPpolypeptide amino acid sequence (SEQ ID NO:1) are: BmIAP cDNA nucleotidesequence:    1 CATTATTAAA CTCACTTCAC TTCGGTAGTG TGAATGTTAA CGTGAAACTCCGCGCTCTTC   61 TTTAGTTGCT ACTCGGTTCT GTCTGGCTGC GTCTGGCTGC TGGAACTTCATACTATTTTG  121 TTCTTGCAAG ACGAGTGTCA GTGATTAAAC AAAAACATAA GAATAGACGTTTTATGCGTT  181 ACTAAAAAAA AGGAAAAATA TACCAATGGA GTTGACGAAA GTTGCTAAAAATGGAGCTGC  241 CGCCACGTTG GTGATGTTAA AAAATGCGCG GGATGCAAAA ATGCGACCTTTCATTGGTCC  301 GCTCATGTTA TCCTCGTGTG AGTCTTCAAC GACATCCACA CTCCCGTCACCTTCGTCGTC  361 AGCTGATAAA ACGGATAATC ACGACACATT CAACTTCCTT CCTGATATGCCCGACATGCG  421 TCGTGAAGAG GAACGTCTGA AAACATTTGA TCAGTGGCCC GTTACGTTTTTGACGCCGGA  481 ACAATTGGCC CGCAACGGAT TCTACTACGT CGGTCGCGGC GACGAAGTGTGCTGTGCTTT  541 CTGTAAGGTA GAAATTATGA GGTGGGTCGA AGGCGACGAT CCTGCCGCCGATCATCGGAG  601 ATGGGCGCGC CAGTGTCCCT TTGTACGAAA ACAAATGTAT GCCAACGCTGGGGGAGAGGC  661 GACCGCTGTC GGTAGAGAGG AATGTGGGGC CAGTGCGGCC ACGCAGCCTCCCCGCATGCC  721 CGGCCCCGTG CACGCGCGGT ACTCCACCGA GGCCGCGCGG CTCGCCACCTTCAAGGACTG  781 GCCGAGACGT ATGCGCCAAA AACCCGAGGA ACTGGCAGAG GCGGGATTCTTCTATACAGG  841 CCAAGGTGAC AAAACGAAAT GCTTCTATTG CGACGGAGGG CTAAAAGATTGGGAAAGCGA  901 TGACGTTCCG TGGGAACAGC ACGCCAGATG GTTCGACCGC TGCGCGTACGTGCAATTGGT  961 GAAAGGACGT GACTACATTC AGAAGGTGAA GTCGGAGGCC ACTGCGATATCTGCTAGCGA 1021 AGAAGAACAG GCCGCCACCA ATGATTCGAC TAAGAACGTC GCCCAAGAGGGCGAGAAACA 1081 TTTGGATGAC TCTAAAATAT GTAAAATATG TTATTCCGAG GAGCGTAACGTGTGCTTCGT 1141 GCCGTGCGGC CACGTGGTGG CGTGCGCCAA GTGCGCGCTG TCGACGGACAAGTGCCCGAT 1201 GTGTCGCAGG ACGTTCACGA ATGCGGTGCG GCTCTACTTC TCGTGAAAGGACCCTCCTCG 1261 CGAGCTGTAT ACTAATCACT TCACCGGGCG GCCCTGGAGC GTGCTGAAACCACGCTTCGA 1321 ACGAAACCGC GTATCCTGTG ATTTTTACAT TAAATAAATT TACAAATTGATAGCGGTGGG 1381 GCAATGTATA GGAACTCGTC AGAACTCGCG AGTTGACGTG CAGGAAGGAGTTAGTGATTT 1441 GTAAACTTGT AAACTGATGT TGAAATGATT TTATTTATTA TTTAAAATTCTAATGACAAA 1501 GTGTAAGTAA ATAAATGTAC ATATTATTTT AGATTATCAG TTTGTCCCACCGACAAAAGT 1561 GAAATGTACA TAGGTGTTTT CATATCACTT CAACAGTCGA AGACCTTCTTTTTGAATTTA 1621 AGGATATATA TTTATACATA TAAATTAAAA TTTTAACGAG AGATCAATATAAATGGTTTA 1681 ACAACTTATT TATACACTGA AATCAAGTGA AGTGTAACAT GGTCTGAAGAATGTTTTACT 1741 GATTTCACTT CCCCTGTTGA AGTGATAAAA TTCTAATGTA AATCCAGAGTTTAAATGTCG 1801 TCATAATTAA TATAAGAAAC AAGTTTTACG CTTCTTTTGC TTGAAAAATCTTATAATTGA 1861 TTCAGGAATT ATTTAATGTG ACTATATTTT GTTCCTGTAA ATAACATAATATATACTATT 1921 TATTGATTAA TTCTGACATA ATTTATGGCA ATTCCGTAAG ATACAATCCAATACTTATTT 1981 CATGTAACTC ACTTCAAAAT AGTTGAATGT GTGGTGTGAT TATAATGTTAAATGTCTAAA 2041 TTTATAATAA ATTGAGCAAA GTTGCATTTA ATGTATGAAT ACTAATTATTGTTTTAACAA 2101 AACATTTAAG TATAATGTGC TCTGTGATTT TAATGTATCA AGAAATAAGCCCAACACCTT 2161 AATTGAAGTT TTTACATTGT TGCTGATAAA AAAAATCATA TCAATTACATTTACAAGTCA 2221 ATTTTAATTG TTCAGAAACC AAACACAATT TTGTTAGTGA CTCCTGCTTTACGAAGTAGT 2281 ATGACAAACC AGTGTTTCGT TGATTGCATT AATTTAGTTG TAACCAATATTTACACTCAA 2341 CATTTTAAGA TGTCATTGAG GAATTCTGTA TAAAAAATGG GAATTTATTTATTGGTGTAT 2401 AATACAATCG CGCACAAGCC ATTTGCAAGT TTCTACACAA CTAAAACGTATTGTATCCAT 2461 TATCTATACG TCATATCATT AATATATACT TGCTTTAGCA AACATATATTCACGAATAAC 2521 TTCACAATAT ATTTTTGTAA ATCAACATAT TAATGGTAAT TAACGAATCGCACGGTACAA 2581 ATAGTGATAA CTGCTGAGTG CACTAAATAG TAAGAGAATT TATTTAAACAGTCAAATTTT 2641 GTTTCATAAG TAGTTATTTC ATACTGTTGA ATGTTATTCA TTAAAACAAATGTTAAAGCA 2701 AAAAAAAAAA AAAAAAGTCG TGACTGGGAA AA (SEQ ID NO:1) BmIAPcoding region nucleotide sequence:    1 ATGGAGTTGA CGAAAGTTGC TAAAAATGGAGCTGCCGCCA CGTTGGTGAT GTTAAAAAAT   61 GCGCGGGATG CAAAAATGCG ACCTTTCATTGGTCCGCTCA TGTTATCCTC GTGTGAGTCT  121 TCAACGACAT GCACACTCCC GTCACCTTCGTCGTCAGCTG ATAAAACGGA TAATCACGAC  181 ACATTCAACT TCCTTCCTGA TATGCCCGACATGCGTCGTG AAGAGGAACG TCTGAAAACA  241 TTTGATCAGT GGCCCGTTAC GTTTTTGACGCCGGAACAAT TGGCCCGCAA CGGATTCTAC  301 TACGTGGGTC GCGGCGACGA AGTGTGCTGTGCTTTCTGTA AGGTAGAAAT TATGAGGTGG  361 GTCGAAGGCG ACGATCCTGC CGCCGATCATCGGAGATGGG CGGCCCAGTG TCCCTTTGTA  421 CGAAAACAAA TGTATGCCAA CGCTGGGGGAGAGGCGACCG CTGTCGGTAG AGACGAATGT  481 GGGGCCAGTG CGGCCACGCA GCCTCCCCGCATGCCCGGCC CCGTGCACGC GCGGTACTGC  541 ACCGAGGCCG CGCGGCTCGC CACCTTCAAGGACTGGCCGA GACGTATGCG CCAAAAACCC  601 GAGGAACTGG CAGAGGCCGG ATTCTTCTATACAGGCCAAG GTGACAAAAC GAAATGCTTC  661 TATTGCGACG GAGGGCTAAA AGATTGGGAAAGCGATGACG TTCCGTGGGA ACAGCACGCC  721 AGATGGTTCG ACCGCTGCGC GTACGTGCAATTGGTGAAAG GACGTGACTA CATTCAGAAG  781 GTGAAGTCGG AGGCCACTGC GATATCTGCTAGCGAAGAAG AACAGGCCGC CACCAATGAT  841 TCGACTAAGA ACGTCGCCCA AGAGGGCGAGAAACATTTGG ATGACTCTAA AATATGTAAA  901 ATATGTTATT CCGAGGAGCG TAACGTGTGCTTCGTGCCGT GCGGCCACGT GGTGGCGTGG  961 GCCAAGTGCG CGCTGTCGAC GGACAAGTGCCCGATGTGTC GCAGGACGTT CACGAATGCG 1021 GTGCGGCTCT ACTTCTCGTG A BmIAPpolypeptide amino acid sequence: (SEQ ID NO:2)    1 M E L T K V A K N GA A A T L V M L K N A R D A K M R P F I   31 G P L M L S S C E S S T T ST L P S P S S S A D K T D N H D   61 T F N F L P D M P D M R R E E E R LK T F D Q W P V T F L T   91 P E Q L A R N G F Y Y L G R G D E V C C A FC K V E T M R W  121 V E G D D P A A D H R R W A P Q C P F V R K Q M Y AN A G G  151 E A T A V G R D E C G A S A A T Q P P R M P G P V H A R Y S 181 T E A A R L A T F K D W P R R M R Q K P E E L A E A G F F Y  211 TG Q G D K T K C F Y C D G G L K D W E S D D V P W E Q H A  241 R W F D RC A Y V Q L V K G R D Y I Q K V K S E A T A I S A  271 S E E E Q A A T ND S T K N V A Q E G E K H L D D S K I C K  301 I C Y S E E R N V C F V PC G H V V A C A K C A L S T D K C  331 P M C R R T F T N A V R L Y F S *

[0095] The full-length BmIAP cDNA (SEQ ID NO:1) (Genbank accessionnumber AF281073) contains a continuous open reading frame (ORF) encodinga protein of 346 amino acids (FIG. 1B). This ORF is initiated by an AUGwithin a favorable context for translation (see, e.g., Kozak (1996)Mammalian Genomes 7:563-574) and is preceded by upstream stop codons inall three reading frames. FIG. 1A shows the location of the BIR domains(BIR1, residues 74 to 140; BIR2, residues 182 to 249; of SEQ ID NO:2)and the RING domain (residues 298 to 314; of SEQ ID NO:2) of BmIAP. FIG.1B is the full length amino acid sequence of BmIAP (SEQ ID NO:2).Sequence alignments of the BIR1 (FIG. 1C), BIR2 (FIG. 1D) and RING (FIG.1E) domains of BmIAP with the corresponding domains of other IAP familymembers are shown, with bold text indicates identical amino acid. TheGenbank accession numbers of sequences used for the alignments are:Bombyx mori IAP (BmIAP) AF281073, Spodoptera frugiperda IAP (SfIAP)AF186378, Trichoplusia ni IAP (TnIAP) AF195528, Orgyia pseudotsugatanucleopolyhedrovirus IAP (OpIAP) P41437, Cydia pomonella granulovirusIAP (CpIAP) P41436, and Drosophila melanogaster IAP1 (DIAP1) Q24306.

[0096] Similar to SfIAP, the BmIAP protein contains two BIR domainsfollowed by a RING domain near its C-terminus (FIG. 1A and 1B). In theBIR domain of BmIAP, the conserved presence and spacing of cysteine andhistidine residue (CX₂CX₆WX₉HX₆C) (SEQ ID NO: 7) is also observed.Within BIR and RING regions, BmIAP shares 88% amino acid identity (92%similarity) with SfIAP, 90% identity (92% similarity) with TnIAP and 76%identity (81% similarity) with CpIAP (FIG. 1C-1E). Thus BmIAP shareshigh sequence sequence similarity with the other two lepidopteran IAPs,SfIAP and TnIAP, suggesting evolutionary conservation.

[0097] Experiments demonstrated that both BIR and RING domains of BmIAPare required to block apoptosis induced by apoptosis suppressorp35-deficient AcMNPV virus. The anti-apoptotic activity of BmIAP ininsect cells was tested by co-transfecting AcMNPV p35-deficient viralDNA into Sf-21 cells with plasmids containing full-length BmIAP (SEQ IDNO:2) or truncation mutants of BmIAP lacking two BIR domains or the RINGdomain (see FIG. 1A). Cells expressing IAPs/IAP fragments withanti-apoptotic activity support virus replication, whereas cells withouta functional IAP are unable to support virus replication and undergoapoptosis. Production of viable viral progeny from rescued virusesresults in the formation of occlusion bodies, serving as a convenientend-point. Occlusion body formation serves as a visual screeningend-point under light-microscopy, as described by Crook (1993) J. ofVirol. 67:2168-2174; Birnbaum (1994) J. of Virol. 68:2521-2525.Sf-21cells co-transfected with p35-deficient AcMNPV viral DNA and (FIG. 2A)BmIAP, or (FIG. 2D) SfIAP, show the production of occlusion bodies asindicated by arrowheads, whereas (FIG. 2B) BmIAP-BIR, (FIG. 2C)BmIAP-RING, and (FIG. 2E) AcIAP show apoptotic body formation (indicatedby arrowheads) without occlusion body formation. Since SfIAP has beenshown to block apoptosis in both insect and mammalian cells (see, e.g.,Huang (2000) supra), whereas AcIAP is ineffective (Clem (1994) Mol.Cell. Biol. 14:5212-5222; Huang (2000) supra, SfIAP and AcIAP were usedas positive and negative controls, respectively (FIG. 2D and E). FIG. 2Fis the mock transfection control.

[0098] A plasmid encoding the full-length BmIAP (SEQ ID NO:2) was ableto complement the p35 (apoptosis inhibiting)-deficiency in thebaculovirus, supporting occlusion body production and virus replicationat 3 days post-transfection (FIG. 2A). In contrast, neither the BIR norRING domain deletion mutants of BmIAP (see FIG. 1A) was able to supportocclusion body formation (thus, no apoptosis inhibiting activity). Sf-21cells displayed morphological changes of apoptosis, such as apoptoticbody formation, when either the BIR or RING domain deletion mutants wereco-transfected with p35-deficient viral DNA (FIG. 2B and C). Theseresults demonstrate that both the BIR and RING domain regions of BmIAP(BIR1, residues 74 to 140; BIR2, residues 182 to 249; of SEQ ID NO:2)and the RING domain (residues 298 to 314; of SEQ ID NO:2) are requiredin combination for the anti-apoptotic function in insect cells.

[0099] Experiments demonstrated that BmIAP inhibited Bax-induced but notFas-induced apoptosis in mammalian cells (i.e., BniIAP protectsmammalian cells against Bax-induced but not Fas-induced apoptosis).Expression plasmid encoding Bax (FIG. 3A) or Fas (FIG. 3B) wereco-transfected into HEK 293 cells with the indicated myc-tagged IAPexpression plasmids. Percentage apoptosis was measured 24 to 36 hourspost-transfection by 4′-6-diamidino-2-phenylindole (DAPI) staining (mean+/−S.D., n=3). Recombinant BmIAP (2 uM) was added to cytosolic extracts(10 mg/ml) from HEK293 cells concurrently with the addition of 1 uMcytochrome-c/10 mM dATP. After incubation at 30° C. for 10 minutes,aliquots were withdrawn and assayed for caspase activity, as measured byrelease of AFC from Ac-DEVD-AFC substrate (100 uM). Data are presentedin FIG. 3C as a percentage relative to control reaction in whichcytochrome-c/dATP were added alone.

[0100] SfIAP and baculoviral IAPs were previously shown to blockapoptosis in mammalian cells (Huang (2000) supra; Hawkins (1996) Proc.Natl. Acad. Sci. USA 93:13786-13790; Uren (1996) Proc. Natl. Acad. Sci.USA 93:4974-4978; Hawkins (1998) Cell Death and Differentiation5:569-576). To explore whether BmIAP has similar properties, weco-expressed BmIAP in HEK293 cells with either Fas or Bax, representingtwo major pathways that utilize caspase-8 and caspase-9, respectively,as their apical proteases. Similar to SfIAP, full-length BmIAP (SEQ IDNO:2) inhibited Bax (FIG. 3A) but not Fas-induced apoptosis (FIG. 3B).In contrast human XIAP protected cells against both Bax and Fas-inducedapoptosis. As in Sf-21 cells, the inhibition of Bax-induced apoptosis inmammalian cells also requires both the BIR and RING domains of BmIAP(SEQ ID NO:2) (see FIG. 1A), suggesting the conservation of thestructural requirements for inhibition (FIG. 3A). Immunoblot analysisindicated that the levels of the BIR and RING truncation proteins weresimilar to that of full-length BmIAP in transfected cells, excludingdifferences in protein levels as an explanation for the failure of theBIR domains or RING domain to suppress cell death.

[0101] These results were further confirmed in a cell-free system inwhich exogenously added cytochrome-c, an agonist of the caspase-9activating protein Apaf-1 (see, e.g., Zou (1997) Cell 90:405-413),induced activation of caspase-3 and similar effector proteases. BmIAPdirectly suppressed caspase-9, but not caspase-3 or caspase-7. This wasmeasured by hydrolysis of Ac-DEVD-AFC, as described by Quan (1995) J.Biol. Chem. 270:10377-10379). Recombinant active (FIG. 4A) caspase-9 wasadded at 0.2 uM and incubated at 37° C. with Ac-LEHD-AFC substrate (100uM) in the presence or absence of various concentration (0.2-1.6 uM) ofrecombinant purified BmIAP or SfIAP. AFC release was measuredcontinuously. In FIG. 4, data are expressed as a percentage relative tocontrol reactions lacking IAPs, using rates determined from the linearportion of enzyme progress curves. Various control GST-fusion proteinshad no inhibition effect.

[0102] Recombinant caspase-3 (2.6 nM) was incubated at 37° C. withAc-DEVD-AFC substrate (100 uM) in the presence or absence of 0.05 uMGST-XIAP, 0.5 uM GST-BmIAP (200 fold molar excess to caspase) or 0.5 uMGST-SfIAP (200 molar excess) (FIG. 4B). AFC release was measured asabove.

[0103] Recombinant caspase-7 (7.0 nM) was incubated at 37° C. withAc-DEVD-AFC substrate (100 uM) in the presence or absence of 0.14 uMGST-XIAP, 0.7 uM GST-BmIAP (100 fold molar excess relative to caspase or0.7 uM GST-SfIAP (100 molar excess) (FIG. 4C). AFC release was measuredas above. In cytosolic extracts treated with cytochrome-c, recombinantBmIAP and positive control recombinant SfIAP completely blocked thehydrolysis of Ac-DEVD-AFC whereas negative control recombinant XIAP-BIR1had no effect on caspase activity (FIG. 4C). Since caspases-3 and -7 arecommon to both Bax and Fas pathways, these results demonstrate thatBmIAP, like SfIAP, inhibits the mitochondria/cytochrome-c pathway inmammalian cells, thus, suppressing apoptosis at a step upstream ofcaspases-3 and -7. This finding is supported by the observation thatBmIAP does not inhibit caspases-3 and -7 in vitro.

[0104] These experiments demonstrated that BmIAP is a direct inhibitorof caspase-9. Purified recombinant BmIAP was incubated with purifiedrecombinant caspase-9. Residual activity was measured using Ac-LEHD-AFCas a substrate of caspase-9. BmIAP inhibited recombinant caspase-9 in aconcentration-dependent manner. The relative amount of BmIAP requiredfor caspase-9 inhibition was about 8 fold molar excess (FIG. 4A),similar to the results reported previous for SfIAP and XIAP (Deveraux(1999) supra; Huang (2000) supra; SfIAP was shown to directly inhibitcaspase-9). Unlike XIAP, but similar to SfIAP, BmIAP did not inhibitrecombinant caspase-3 and caspase-7 (caspases-3, -7 and -9 are involvedin apoptotic pathway induced by Bax), suggesting a narrower range ofcaspases specificity compared to human XIAP (FIG. 4B and C).

[0105] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1 25 1 3773 DNA Bombyx mori CDS (2733)..(3770) 1 cattattaaa ctcacttcacttcggtagtg tgaatgttaa cgtgaaactc cgcgctcttc 60 tttagttgct actcggttctgtctggctgc gttgacgttt tggaacttca tactattttg 120 ttcttgcaag acgagtgtcagtgattaaac aaaaacataa gaatagacgt tttatgcgtt 180 actaaaaaaa aggaaaaatataccaatgga gttgacgaaa gttgctaaaa atggagctgc 240 cgccacgttg gtgatgttaaaaaatgcgcg ggatgcaaaa atgcgacctt tcattggtcc 300 gctcatgtta tcctcgtgtgagtcttcaac gacatccaca ctcccgtcac cttcgtcgtc 360 agctgataaa acggataatcacgacacatt caacttcctt cctgatatgc ccgacatgcg 420 tcgtgaagag gaacgtctgaaaacatttga tcagtggccc gttacgtttt tgacgccgga 480 acaattggcc cgcaacggattctactacct cggtcgcggc gacgaagtgt gctgtgcttt 540 ctgtaaggta gaaattatgaggtgggtcga aggcgacgat cctgccgccg atcatcggag 600 atgggcgccc cagtgtccctttgtacgaaa acaaatgtat gccaacgctg ggggagaggc 660 gaccgctgtc ggtagagacgaatgtggggc cagtgcggcc acgcagcctc cccgcatgcc 720 cggccccgtg cacgcgcggtactccaccga ggccgcgcgg ctcgccacct tcaaggactg 780 gccgagacgt atgcgccaaaaacccgagga actggcagag gccggattct tctatacagg 840 ccaaggtgac aaaacgaaatgcttctattg cgacggaggg ctaaaagatt gggaaagcga 900 tgacgttccg tgggaacagcacgccagatg gttcgaccgc tgcgcgtacg tgcaattggt 960 gaaaggacgt gactacattcagaaggtgaa gtcggaggcc actgcgatat ctgctagcga 1020 agaagaacag gccgccaccaatgattcgac taagaacgtc gcccaagagg gcgagaaaca 1080 tttggatgac tctaaaatatgtaaaatatg ttattccgag gagcgtaacg tgtgcttcgt 1140 gccgtgcggc cacgtggtggcgtgcgccaa gtgcgcgctg tcgacggaca agtgcccgat 1200 gtgtcgcagg acgttcacgaatgcggtgcg gctctacttc tcgtgaaagg accctcctcg 1260 cgagctgtat actaatcacttcaccgggcg gccctggagc gtgctgaaac cacccttcga 1320 acgaaaccgc gtatcctgtgatttttacat taaataaatt tacaaattga tagcggtggg 1380 gcaatgtata ggaactcgtcagaactcgcg agttgacgtg caggaaggag ttagtgattt 1440 gtaaacttgt aaactgatgttgaaatgatt ttatttatta tttaaaattc taatgacaaa 1500 gtgtaagtaa ataaatgtacatattatttt agattatcag tttgtcccac cgacaaaagt 1560 gaaatgtaca taggtgttttcatatcactt caacagtcga agaccttctt tttgaattta 1620 aggatatata tttatacatataaattaaaa ttttaacgag acatcaatat aaatggttta 1680 acaacttatt tatacactgaaatcaagtga agtgtaacat ggtctgaaga atgttttact 1740 gatttcactt cccctgttgaagtgataaaa ttctaatgta aatccagagt ttaaatgtcg 1800 tcataattaa tataagaaacaagttttacg cttcttttgc ttgaaaaatc ttataattga 1860 ttcaggaatt atttaatgtgactatatttt gttcctgtaa ataacataat atatactatt 1920 tattgattaa ttctgacataatttatggca attccgtaag atacaatcca atacttattt 1980 catgtaactc acttcaaaatagttgaatgt gtggtgtgat tataatgtta aatgtctaaa 2040 tttataataa attgagcaaagttgcattta atgtatgaat actaattatt gttttaacaa 2100 aacatttaag tataatctgctctgtgattt taatgtatca agaaataacc ccaacacctt 2160 aattgaagtt tttacattgttgctgataaa aaaaatcata tcaattacat ttacaagtca 2220 attttaattg ttcagaaaccaaacacaatt ttgttagtga ctcctgcttt acgaagtagt 2280 atgacaaacc agtgtttcgttgattgcatt aatttagttg taaccaatat ttacactcaa 2340 cattttaaga tgtcattgaggaattctgta taaaaaatgg gaatttattt attggtgtat 2400 aatacaatcc cgcacaagccatttgcaagt ttctacacaa ctaaaacgta ttgtatccat 2460 tatctatacg tcatatcattaatatatact tgctttagca aacatatatt cacgaataac 2520 ttcacaatat atttttgtaaatcaacatat taatggtaat taacgaatcg cacggtacaa 2580 atagtgataa ctgctgagtgcactaaatag taagagaatt tatttaaaca gtcaaatttt 2640 gtttcataag tagttatttcatactgttga atgttattca ttaaaacaaa tgttaaagca 2700 aaaaaaaaaa aaaaaagtcgtgactgggaa aa atg gag ttg acg aaa gtt gct 2753 Met Glu Leu Thr Lys ValAla 1 5 aaa aat gga gct gcc gcc acg ttg gtg atg tta aaa aat gcg cgg gat2801 Lys Asn Gly Ala Ala Ala Thr Leu Val Met Leu Lys Asn Ala Arg Asp 1015 20 gca aaa atg cga cct ttc att ggt ccg ctc atg tta tcc tcg tgt gag2849 Ala Lys Met Arg Pro Phe Ile Gly Pro Leu Met Leu Ser Ser Cys Glu 2530 35 tct tca acg aca tcc aca ctc ccg tca cct tcg tcg tca gct gat aaa2897 Ser Ser Thr Thr Ser Thr Leu Pro Ser Pro Ser Ser Ser Ala Asp Lys 4045 50 55 acg gat aat cac gac aca ttc aac ttc ctt cct gat atg ccc gac atg2945 Thr Asp Asn His Asp Thr Phe Asn Phe Leu Pro Asp Met Pro Asp Met 6065 70 cgt cgt gaa gag gaa cgt ctg aaa aca ttt gat cag tgg ccc gtt acg2993 Arg Arg Glu Glu Glu Arg Leu Lys Thr Phe Asp Gln Trp Pro Val Thr 7580 85 ttt ttg acg ccg gaa caa ttg gcc cgc aac gga ttc tac tac ctc ggt3041 Phe Leu Thr Pro Glu Gln Leu Ala Arg Asn Gly Phe Tyr Tyr Leu Gly 9095 100 cgc ggc gac gaa gtg tgc tgt gct ttc tgt aag gta gaa att atg agg3089 Arg Gly Asp Glu Val Cys Cys Ala Phe Cys Lys Val Glu Ile Met Arg 105110 115 tgg gtc gaa ggc gac gat cct gcc gcc gat cat cgg aga tgg gcg ccc3137 Trp Val Glu Gly Asp Asp Pro Ala Ala Asp His Arg Arg Trp Ala Pro 120125 130 135 cag tgt ccc ttt gta cga aaa caa atg tat gcc aac gct ggg ggagag 3185 Gln Cys Pro Phe Val Arg Lys Gln Met Tyr Ala Asn Ala Gly Gly Glu140 145 150 gcg acc gct gtc ggt aga gac gaa tgt ggg gcc agt gcg gcc acgcag 3233 Ala Thr Ala Val Gly Arg Asp Glu Cys Gly Ala Ser Ala Ala Thr Gln155 160 165 cct ccc cgc atg ccc ggc ccc gtg cac gcg cgg tac tcc acc gaggcc 3281 Pro Pro Arg Met Pro Gly Pro Val His Ala Arg Tyr Ser Thr Glu Ala170 175 180 gcg cgg ctc gcc acc ttc aag gac tgg ccg aga cgt atg cgc caaaaa 3329 Ala Arg Leu Ala Thr Phe Lys Asp Trp Pro Arg Arg Met Arg Gln Lys185 190 195 ccc gag gaa ctg gca gag gcc gga ttc ttc tat aca ggc caa ggtgac 3377 Pro Glu Glu Leu Ala Glu Ala Gly Phe Phe Tyr Thr Gly Gln Gly Asp200 205 210 215 aaa acg aaa tgc ttc tat tgc gac gga ggg cta aaa gat tgggaa agc 3425 Lys Thr Lys Cys Phe Tyr Cys Asp Gly Gly Leu Lys Asp Trp GluSer 220 225 230 gat gac gtt ccg tgg gaa cag cac gcc aga tgg ttc gac cgctgc gcg 3473 Asp Asp Val Pro Trp Glu Gln His Ala Arg Trp Phe Asp Arg CysAla 235 240 245 tac gtg caa ttg gtg aaa gga cgt gac tac att cag aag gtgaag tcg 3521 Tyr Val Gln Leu Val Lys Gly Arg Asp Tyr Ile Gln Lys Val LysSer 250 255 260 gag gcc act gcg ata tct gct agc gaa gaa gaa cag gcc gccacc aat 3569 Glu Ala Thr Ala Ile Ser Ala Ser Glu Glu Glu Gln Ala Ala ThrAsn 265 270 275 gat tcg act aag aac gtc gcc caa gag ggc gag aaa cat ttggat gac 3617 Asp Ser Thr Lys Asn Val Ala Gln Glu Gly Glu Lys His Leu AspAsp 280 285 290 295 tct aaa ata tgt aaa ata tgt tat tcc gag gag cgt aacgtg tgc ttc 3665 Ser Lys Ile Cys Lys Ile Cys Tyr Ser Glu Glu Arg Asn ValCys Phe 300 305 310 gtg ccg tgc ggc cac gtg gtg gcg tgc gcc aag tgc gcgctg tcg acg 3713 Val Pro Cys Gly His Val Val Ala Cys Ala Lys Cys Ala LeuSer Thr 315 320 325 gac aag tgc ccg atg tgt cgc agg acg ttc acg aat gcggtg cgg ctc 3761 Asp Lys Cys Pro Met Cys Arg Arg Thr Phe Thr Asn Ala ValArg Leu 330 335 340 tac ttc tcg tga 3773 Tyr Phe Ser 345 2 346 PRTBombyx mori 2 Met Glu Leu Thr Lys Val Ala Lys Asn Gly Ala Ala Ala ThrLeu Val 1 5 10 15 Met Leu Lys Asn Ala Arg Asp Ala Lys Met Arg Pro PheIle Gly Pro 20 25 30 Leu Met Leu Ser Ser Cys Glu Ser Ser Thr Thr Ser ThrLeu Pro Ser 35 40 45 Pro Ser Ser Ser Ala Asp Lys Thr Asp Asn His Asp ThrPhe Asn Phe 50 55 60 Leu Pro Asp Met Pro Asp Met Arg Arg Glu Glu Glu ArgLeu Lys Thr 65 70 75 80 Phe Asp Gln Trp Pro Val Thr Phe Leu Thr Pro GluGln Leu Ala Arg 85 90 95 Asn Gly Phe Tyr Tyr Leu Gly Arg Gly Asp Glu ValCys Cys Ala Phe 100 105 110 Cys Lys Val Glu Ile Met Arg Trp Val Glu GlyAsp Asp Pro Ala Ala 115 120 125 Asp His Arg Arg Trp Ala Pro Gln Cys ProPhe Val Arg Lys Gln Met 130 135 140 Tyr Ala Asn Ala Gly Gly Glu Ala ThrAla Val Gly Arg Asp Glu Cys 145 150 155 160 Gly Ala Ser Ala Ala Thr GlnPro Pro Arg Met Pro Gly Pro Val His 165 170 175 Ala Arg Tyr Ser Thr GluAla Ala Arg Leu Ala Thr Phe Lys Asp Trp 180 185 190 Pro Arg Arg Met ArgGln Lys Pro Glu Glu Leu Ala Glu Ala Gly Phe 195 200 205 Phe Tyr Thr GlyGln Gly Asp Lys Thr Lys Cys Phe Tyr Cys Asp Gly 210 215 220 Gly Leu LysAsp Trp Glu Ser Asp Asp Val Pro Trp Glu Gln His Ala 225 230 235 240 ArgTrp Phe Asp Arg Cys Ala Tyr Val Gln Leu Val Lys Gly Arg Asp 245 250 255Tyr Ile Gln Lys Val Lys Ser Glu Ala Thr Ala Ile Ser Ala Ser Glu 260 265270 Glu Glu Gln Ala Ala Thr Asn Asp Ser Thr Lys Asn Val Ala Gln Glu 275280 285 Gly Glu Lys His Leu Asp Asp Ser Lys Ile Cys Lys Ile Cys Tyr Ser290 295 300 Glu Glu Arg Asn Val Cys Phe Val Pro Cys Gly His Val Val AlaCys 305 310 315 320 Ala Lys Cys Ala Leu Ser Thr Asp Lys Cys Pro Met CysArg Arg Thr 325 330 335 Phe Thr Asn Ala Val Arg Leu Tyr Phe Ser 340 3453 20 DNA Artificial Sequence Description of Artificial Sequence Primer 3gcngangcng gnttyttyta 20 4 17 DNA Artificial Sequence Description ofArtificial Sequence Primer 4 acnacrtgnc crcangg 17 5 18 DNA ArtificialSequence Description of Artificial Sequence Primer 5 ctgttcccac ggaacgtc18 6 17 DNA Artificial Sequence Description of Artificial SequencePrimer 6 gccaccaatg attcgac 17 7 28 PRT Artificial Sequence Descriptionof Artificial Sequence Conserved motif 7 Cys Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Xaa Trp Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa His Xaa XaaXaa Xaa Xaa Xaa Cys 20 25 8 172 PRT Bombyx mori 8 Glu Glu Glu Arg LeuLys Thr Phe Asp Gln Trp Pro Val Thr Phe Leu 1 5 10 15 Thr Pro Glu GlnLeu Ala Arg Asn Gly Phe Tyr Tyr Leu Gly Arg Gly 20 25 30 Asp Glu Val CysCys Ala Phe Cys Lys Val Glu Ile Met Arg Trp Val 35 40 45 Glu Gly Asp AspPro Ala Ala Asp His Arg Arg Trp Ala Pro Gln Cys 50 55 60 Pro Phe Val GluAla Ala Arg Leu Ala Thr Phe Lys Asp Trp Pro Arg 65 70 75 80 Arg Met ArgGln Lys Pro Glu Glu Leu Ala Glu Ala Gly Phe Phe Tyr 85 90 95 Thr Gly GlnGly Asp Lys Thr Lys Cys Phe Tyr Cys Asp Gly Gly Leu 100 105 110 Lys AspTrp Glu Ser Asp Asp Val Pro Trp Glu Gln His Ala Arg Trp 115 120 125 PheAsp Arg Cys Ala Tyr Val Leu Cys Lys Ile Cys Tyr Ser Glu Glu 130 135 140Arg Asn Val Cys Phe Val Pro Cys Gly His Val Val Ala Cys Ala Lys 145 150155 160 Cys Ala Leu Ser Thr Asp Lys Cys Pro Met Cys Arg 165 170 9 172PRT Spodoptera frugiperda 9 Glu Asp Glu Arg Met Lys Thr Phe Glu Lys TrpPro Val Ser Phe Leu 1 5 10 15 Ser Gly Glu Gln Leu Ala Arg Asn Gly PheTyr Tyr Leu Gly Arg Arg 20 25 30 Asp Glu Ala Arg Cys Ala Phe Cys Lys ValGlu Ile Met Arg Trp Val 35 40 45 Glu Gly Asp Asp Pro Ala Lys Asp His GlnArg Trp Ala Pro Gln Cys 50 55 60 Pro Phe Val Glu Ala Ala Arg Leu Arg SerPhe Lys Asp Trp Pro Arg 65 70 75 80 Cys Met Arg Gln Lys Pro Glu Glu LeuAla Glu Ala Gly Phe Phe Tyr 85 90 95 Thr Gly Gln Gly Asp Lys Thr Lys CysPhe Tyr Cys Asp Gly Gly Leu 100 105 110 Lys Asp Trp Glu Asn His Asp ValPro Trp Glu Gln His Ala Arg Trp 115 120 125 Phe Asp Arg Cys Ala Tyr ValLeu Cys Lys Ile Cys Tyr Ala Glu Glu 130 135 140 Arg Asn Val Cys Phe ValPro Cys Gly His Val Val Ala Cys Ala Lys 145 150 155 160 Cys Ala Leu AlaAla Asp Lys Cys Pro Met Cys Arg 165 170 10 172 PRT Trichoplusia ni 10Glu Asp Glu Arg Ile Lys Thr Phe Glu Lys Trp Pro Val Ser Phe Leu 1 5 1015 Ser Gly Glu Gln Leu Ala Arg Asn Gly Phe Tyr Tyr Leu Gly Arg Gly 20 2530 Asp Glu Val Arg Cys Ala Phe Cys Lys Val Glu Ile Met Arg Trp Val 35 4045 Glu Gly Asp Asp Pro Ala Lys Asp His Gln Arg Trp Ala Pro Gln Cys 50 5560 Pro Phe Val Glu Ala Ala Arg Leu Arg Ser Phe Lys Asp Trp Pro Arg 65 7075 80 Cys Met Arg Gln Lys Pro Glu Glu Leu Ala Glu Ala Gly Phe Phe Tyr 8590 95 Thr Gly Gln Gly Asp Lys Thr Lys Cys Phe Tyr Cys Asp Gly Gly Leu100 105 110 Lys Asp Trp Glu Asn Asp Asp Val Pro Trp Glu Gln His Ala ArgTrp 115 120 125 Phe Asp Arg Cys Ala Tyr Val Leu Cys Lys Ile Cys Phe AlaGlu Glu 130 135 140 Arg Asn Val Cys Phe Val Pro Cys Gly His Val Val AlaCys Ala Lys 145 150 155 160 Cys Ala Leu Ala Ala Asp Lys Cys Pro Met CysArg 165 170 11 172 PRT Cydia pomonella granulovirus 11 Glu Asp Val ArgLeu Asn Thr Phe Glu Lys Trp Pro Val Ser Phe Leu 1 5 10 15 Ser Pro GluThr Met Ala Lys Asn Gly Phe Tyr Tyr Leu Gly Arg Ser 20 25 30 Asp Glu ValArg Cys Ala Phe Cys Lys Val Glu Ile Met Arg Trp Lys 35 40 45 Glu Gly GluAsp Pro Ala Ala Asp His Lys Lys Trp Ala Pro Gln Cys 50 55 60 Pro Phe ValGlu Ala Ala Arg Val Lys Ser Phe His Asn Trp Pro Arg 65 70 75 80 Cys MetLys Gln Arg Pro Glu Gln Met Ala Asp Ala Gly Phe Phe Tyr 85 90 95 Thr GlyTyr Gly Asp Asn Thr Lys Cys Phe Tyr Cys Asp Gly Gly Leu 100 105 110 LysAsp Trp Glu Pro Glu Asp Val Pro Trp Glu Gln His Val Arg Trp 115 120 125Phe Asp Arg Cys Ala Tyr Val Leu Cys Lys Ile Cys Tyr Val Glu Glu 130 135140 Cys Ile Val Cys Phe Val Pro Cys Gly His Val Val Ala Cys Ala Lys 145150 155 160 Cys Ala Leu Ser Val Asp Lys Cys Pro Met Cys Arg 165 170 12172 PRT Orgyia pseudotsugata 12 Lys Ala Ala Arg Leu Gly Thr Tyr Thr AsnTrp Pro Val Gln Phe Leu 1 5 10 15 Glu Pro Ser Arg Met Ala Ala Ser GlyPhe Tyr Tyr Leu Gly Arg Gly 20 25 30 Asp Glu Val Arg Cys Ala Phe Cys LysVal Glu Ile Thr Asn Trp Val 35 40 45 Arg Gly Asp Asp Pro Glu Thr Asp HisLys Arg Trp Ala Pro Gln Cys 50 55 60 Pro Phe Val Glu Ala Ala Arg Leu ArgThr Phe Ala Glu Trp Pro Arg 65 70 75 80 Gly Leu Lys Gln Arg Pro Glu GluLeu Ala Glu Ala Gly Phe Phe Tyr 85 90 95 Thr Gly Gln Gly Asp Lys Thr ArgCys Phe Cys Cys Asp Gly Gly Leu 100 105 110 Lys Asp Trp Glu Pro Asp AspAla Pro Trp Gln Gln His Ala Arg Trp 115 120 125 Tyr Asp Arg Cys Glu TyrVal Leu Cys Lys Ile Cys Leu Gly Ala Glu 130 135 140 Lys Thr Val Cys PheVal Pro Cys Gly His Val Val Ala Cys Gly Lys 145 150 155 160 Cys Ala AlaGly Val Thr Thr Cys Pro Val Cys Arg 165 170 13 172 PRT Drosophilamelanogaster 13 Glu Glu Thr Arg Leu Lys Thr Phe Thr Asp Trp Pro Leu AspTrp Leu 1 5 10 15 Asp Lys Arg Gln Leu Ala Gln Thr Gly Met Tyr Phe ThrHis Ala Gly 20 25 30 Asp Lys Val Lys Cys Phe Phe Cys Gly Val Glu Ile GlyCys Trp Glu 35 40 45 Gln Glu Asp Gln Pro Val Pro Glu His Gln Arg Trp SerPro Asn Cys 50 55 60 Pro Leu Leu Glu Thr Ala Arg Leu Arg Thr Phe Glu AlaTrp Pro Arg 65 70 75 80 Asn Leu Lys Gln Lys Pro His Gln Leu Ala Glu AlaGly Phe Phe Tyr 85 90 95 Thr Gly Val Gly Asp Arg Val Arg Cys Phe Ser CysGly Gly Gly Leu 100 105 110 Met Asp Trp Asn Asp Asn Asp Glu Pro Trp GluGln His Ala Leu Trp 115 120 125 Leu Ser Gln Cys Arg Phe Val Leu Cys LysIle Cys Tyr Gly Ala Glu 130 135 140 Tyr Asn Thr Ala Phe Leu Pro Cys GlyHis Val Val Ala Cys Ala Lys 145 150 155 160 Cys Ala Ser Ser Val Thr LysCys Pro Leu Cys Arg 165 170 14 68 PRT Bombyx mori 14 Glu Ala Ala Arg LeuAla Thr Phe Lys Asp Trp Pro Arg Arg Met Arg 1 5 10 15 Gln Lys Pro GluGlu Leu Ala Glu Ala Gly Phe Phe Tyr Thr Gly Gln 20 25 30 Gly Asp Lys ThrLys Cys Phe Tyr Cys Asp Gly Gly Leu Lys Asp Trp 35 40 45 Glu Ser Asp AspVal Pro Trp Glu Gln His Ala Arg Trp Phe Asp Arg 50 55 60 Cys Ala Tyr Val65 15 68 PRT Spodoptera frugiperda 15 Glu Ala Ala Arg Leu Arg Ser PheLys Asp Trp Pro Arg Cys Met Arg 1 5 10 15 Gln Lys Pro Glu Glu Leu AlaGlu Ala Gly Phe Phe Tyr Thr Gly Gln 20 25 30 Gly Asp Lys Thr Lys Cys PheTyr Cys Asp Gly Gly Leu Lys Asp Trp 35 40 45 Glu Asn His Asp Val Pro TrpGlu Gln His Ala Arg Trp Phe Asp Arg 50 55 60 Cys Ala Tyr Val 65 16 68PRT Trichoplusia ni 16 Glu Ala Ala Arg Leu Arg Ser Phe Lys Asp Trp ProArg Cys Met Arg 1 5 10 15 Gln Lys Pro Glu Glu Leu Ala Glu Ala Gly PhePhe Tyr Thr Gly Gln 20 25 30 Gly Asp Lys Thr Lys Cys Phe Tyr Cys Asp GlyGly Leu Lys Asp Trp 35 40 45 Glu Asn Asp Asp Val Pro Trp Glu Gln His AlaArg Trp Phe Asp Arg 50 55 60 Cys Ala Tyr Val 65 17 68 PRT Cydiapomonella granulovirus 17 Glu Ala Ala Arg Val Lys Ser Phe His Asn TrpPro Arg Cys Met Lys 1 5 10 15 Gln Arg Pro Glu Gln Met Ala Asp Ala GlyPhe Phe Tyr Thr Gly Tyr 20 25 30 Gly Asp Asn Thr Lys Cys Phe Tyr Cys AspGly Gly Leu Lys Asp Trp 35 40 45 Glu Pro Glu Asp Val Pro Trp Glu Gln HisVal Arg Trp Phe Asp Arg 50 55 60 Cys Ala Tyr Val 65 18 68 PRT Orgyiapseudotsugata 18 Glu Ala Ala Arg Leu Arg Thr Phe Ala Glu Trp Pro Arg GlyLeu Lys 1 5 10 15 Gln Arg Pro Glu Glu Leu Ala Glu Ala Gly Phe Phe TyrThr Gly Gln 20 25 30 Gly Asp Lys Thr Arg Cys Phe Cys Cys Asp Gly Gly LeuLys Asp Trp 35 40 45 Glu Pro Asp Asp Ala Pro Trp Gln Gln His Ala Arg TrpTyr Asp Arg 50 55 60 Cys Glu Tyr Val 65 19 68 PRT Drosophilamelanogaster 19 Glu Thr Ala Arg Leu Arg Thr Phe Glu Ala Trp Pro Arg AsnLeu Lys 1 5 10 15 Gln Lys Pro His Gln Leu Ala Glu Ala Gly Phe Phe TyrThr Gly Val 20 25 30 Gly Asp Arg Val Arg Cys Phe Ser Cys Gly Gly Gly LeuMet Asp Trp 35 40 45 Asn Asp Asn Asp Glu Pro Trp Glu Gln His Ala Leu TrpLeu Ser Gln 50 55 60 Cys Arg Phe Val 65 20 37 PRT Bombyx mori 20 Leu CysLys Ile Cys Tyr Ser Glu Glu Arg Asn Val Cys Phe Val Pro 1 5 10 15 CysGly His Val Val Ala Cys Ala Lys Cys Ala Leu Ser Thr Asp Lys 20 25 30 CysPro Met Cys Arg 35 21 37 PRT Spodoptera frugiperda 21 Leu Cys Lys IleCys Tyr Ala Glu Glu Arg Asn Val Cys Phe Val Pro 1 5 10 15 Cys Gly HisVal Val Ala Cys Ala Lys Cys Ala Leu Ala Ala Asp Lys 20 25 30 Cys Pro MetCys Arg 35 22 37 PRT Trichoplusia ni 22 Leu Cys Lys Ile Cys Phe Ala GluGlu Arg Asn Val Cys Phe Val Pro 1 5 10 15 Cys Gly His Val Val Ala CysAla Lys Cys Ala Leu Ala Ala Asp Lys 20 25 30 Cys Pro Met Cys Arg 35 2337 PRT Cydia pomonella granulovirus 23 Leu Cys Lys Ile Cys Tyr Val GluGlu Cys Ile Val Cys Phe Val Pro 1 5 10 15 Cys Gly His Val Val Ala CysAla Lys Cys Ala Leu Ser Val Asp Lys 20 25 30 Cys Pro Met Cys Arg 35 2437 PRT Orgyia pseudotsugata 24 Leu Cys Lys Ile Cys Leu Gly Ala Glu LysThr Val Cys Phe Val Pro 1 5 10 15 Cys Gly His Val Val Ala Cys Gly LysCys Ala Ala Gly Val Thr Thr 20 25 30 Cys Pro Val Cys Arg 35 25 37 PRTDrosophila melanogaster 25 Leu Cys Lys Ile Cys Tyr Gly Ala Glu Tyr AsnThr Ala Phe Leu Pro 1 5 10 15 Cys Gly His Val Val Ala Cys Ala Lys CysAla Ser Ser Val Thr Lys 20 25 30 Cys Pro Leu Cys Arg 35

What is claimed is:
 1. An isolated or recombinant nucleic acidcomprising a nucleic acid sequence having at least 95% sequence identityto SEQ ID NO:1.
 2. The isolated or recombinant nucleic acid of claim 1,wherein the nucleic acid encodes a polypeptide capable of inhibitingapoptosis in insect cells.
 3. The isolated or recombinant nucleic acidof claim 1, wherein the nucleic acid encodes a polypeptide capable ofinhibiting apoptosis in Spodoptera frugiperda or Bombyx mori cells. 4.The isolated or recombinant nucleic acid of claim 1, wherein the nucleicacid encodes a polypeptide capable of inhibiting apoptosis in mammaliancells.
 5. The isolated or recombinant nucleic acid of claim 1, whereinthe nucleic acid encodes a polypeptide capable of inhibiting apoptosisin plant cells.
 6. The isolated or recombinant nucleic acid of claim 1,wherein the nucleic acid encodes a polypeptide capable of inhibitingcaspase
 9. 7. An isolated or recombinant nucleic acid encoding apolypeptide having a sequence as set forth in SEQ ID NO:2.
 8. Anisolated or recombinant nucleic acid comprising a nucleic acid sequenceas set forth in SEQ ID NO:1.
 9. An expression cassette comprising atleast one nucleic acid operably linked to a promoter, wherein thenucleic acid comprises a sequence having at least 95% sequence identityto SEQ ID NO:1.
 10. The expression cassette of claim 9, wherein thepromoter is a constitutive or an inducible promoter.
 11. The expressioncassette of claim 9, wherein the promoter is a developmentally regulatedor a tissue specific promoter.
 12. The expression cassette of claim 9,wherein the nucleic acid encodes a polypeptide having a sequence as setforth in SEQ ID NO:2.
 13. A transformed cell comprising a nucleic acidsequence having at least 95% sequence identity to SEQ ID NO:1.
 14. Thetransformed cell of claim 13, wherein the cell is a mammalian cell. 15.The transformed cell of claim 13, wherein the cell is an insect cell.16. The transformed cell of claim 15, wherein the insect cell is aSpodoptera frugiperda cell.
 17. The transformed cell of claim 13,wherein the cell is a plant cell.
 18. The transformed cell of claim 13,wherein the cell is a yeast cell.
 19. The transformed cell of claim 13,wherein the nucleic acid encodes a polypeptide having a sequence as setforth in SEQ ID NO:2.
 20. A non-human transgenic animal comprising anucleic acid sequence having at least 95% sequence identity to SEQ IDNO:1.
 21. The nonhuman transgenic animal of claim 20, wherein the animalis a rat or a mouse.
 22. The nonhuman transgenic animal of claim 20,wherein the nucleic acid encodes a polypeptide having a sequence as setforth in SEQ ID NO:2.
 23. The nonhuman transgenic animal of claim 20,wherein the nucleic acid encodes a polypeptide capable of inhibitingapoptosis.
 24. A transgenic plant comprising a nucleic acid sequencehaving at least 95% sequence identity to SEQ ID NO:1.
 25. The transgenicplant of claim 24, wherein the nucleic acid encodes a polypeptidecapable of inhibiting apoptosis.
 26. The transgenic plant of claim 24,wherein the plant is abiotic or biotic insult resistant.
 27. Thetransgenic plant of claim 26, wherein the biotic insult is induced by aplant pathogen.
 28. The transgenic plant of claim 27, wherein the plantpathogen is a virus, a fungus, a bacteria or a nemotode.
 29. Thetransgenic plant of claim 26, wherein the abiotic insult is induced byhigh moisture, low moisture, salinity, nutrient deficiency, airpollution, high temperature, low temperature, soil toxicity, herbicidesor insecticides.
 30. The transgenic plant of claim 24, wherein at leasta portion of the plant exhibits a decreased level of senescence.
 31. Aseed capable of germinating into a plant having in its genome aheterologous nucleic acid sequence having at least 95% sequence identityto SEQ ID NO:1.
 32. The seed of claim 31, wherein the nucleic acidencodes a polypeptide capable of inhibiting apoptosis in a plant cell.33. An isolated or recombinant polypeptide comprising a sequence havingat least 95% sequence identity to SEQ ID NO:2.
 34. The isolated orrecombinant polypeptide of claim 33, wherein the polypeptide is capableof inhibiting apoptosis in insect cells.
 35. The isolated or recombinantpolypeptide of claim 33, wherein the polypeptide is capable ofinhibiting apoptosis in Bombyx mori cells.
 36. The isolated orrecombinant polypeptide of claim 33, wherein the polypeptide is capableof inhibiting apoptosis in mammalian cells.
 37. The isolated orrecombinant polypeptide of claim 33, wherein the nucleic acid encodes apolypeptide capable of inhibiting apoptosis in plant cells.
 38. Theisolated or recombinant polypeptide of claim 33, wherein the polypeptideis capable of inhibiting caspase
 9. 39. An isolated or recombinantpolypeptide comprising a sequence as set forth in SEQ ID NO:2.
 40. Afusion protein comprising a sequence having at least 95% sequenceidentity to SEQ ID NO:2 and a second domain.
 41. The fusion protein ofclaim 40, wherein the second domain comprises glutathione S-transferase(GST).
 42. An antibody or binding fragment thereof, wherein the antibodyor fragment specifically binds to a polypeptide or an immunogenicfragment thereof, wherein the polypeptide comprises a sequence having atleast 95% sequence identity to SEQ ID NO:2.
 43. An antibody or bindingfragment thereof, wherein the antibody or fragment specifically binds toa protein having an amino acid sequence as set forth in SEQ ID NO:2 oran immunogenic fragment thereof.
 44. An array comprising a nucleic acidcomprising a sequence having at least 95% sequence identity to SEQ IDNO:1.
 45. A method of detecting or isolating a polypeptide, wherein thepolypeptide comprises a sequence having at least 95% sequence identityto SEQ ID NO:2, comprising contacting a biological sample with anantibody as set forth in claim 38 or claim
 39. 46. A method of making arecombinant polypeptide comprising expressing a nucleic acid comprisinga sequence having at least 95% sequence identity to SEQ ID NO:1.
 47. Amethod for inhibiting apoptosis in a cell comprising the followingsteps: (a) providing an isolated or recombinant polypeptide comprising asequence having at least 95% sequence identity to SEQ ID NO:2, whereinthe polypeptide is capable inhibiting apoptosis in the cell, and (a)contacting the polypeptide with the cell in an amount sufficient toinhibit apoptosis in the cell.
 48. A method for inhibiting apoptosis ina cell comprising the following steps: (a) providing an isolated orrecombinant nucleic acid comprising a sequence having at least 95%sequence identity to SEQ ID NO:1, wherein the nucleic acid encodespolypeptide capable of inhibiting apoptosis in the cell, and (b)contacting the nucleic acid with the cell and expressing the nucleicacid to produce an amount of polypeptide sufficient to inhibit apoptosisin the cell.
 49. The method of claim 47 or claim 48, wherein the cell isan insect cell.
 50. The method of claim 49, wherein the insect cell is aBombyx mori cell.
 51. The method of claim 49, wherein the insect cell isa Spodoptera frugiperda cell.
 52. The method of claim 47 or claim 48,wherein the cell is a mammalian cell.
 53. The method of claim 47 orclaim 48, wherein the cell is a plant cell.
 54. A method for identifyingan agent that can modulate the activity of a polypeptide, wherein thepolypeptide comprises a sequence having at least 95% sequence identityto SEQ ID NO:2 and is capable inhibiting a caspase 9 protease,comprising: (a) providing an isolated or recombinant polypeptidecomprising a sequence having at least 95% sequence identity to SEQ IDNO:2 that is capable inhibiting a caspase 9 protease, and a test agent,(b) contacting the caspase 9 protease and polypeptide in the presenceand absence of the test agent; and (c) measuring the ability of thepolypeptide to inhibit the caspase 9 protease in the presence andabsence of the test agent, wherein an increase or decrease in theability of the polypeptide to inhibit the caspase 9 protease in thepresence of the test agent identifies the test agent as a modulator ofthe polypeptide's activity.
 55. A method for identifying an agent thatcan modulate the activity of a polypeptide, wherein the polypeptidecomprises a sequence having at least 95% sequence identity to SEQ IDNO:2 and is capable inhibiting apoptosis in a cell, comprising: (a)contacting a cell expressing the polypeptide recombinantly in thepresence and absence of a test agent before, during or after inducingapoptosis in the cell; and (b) measuring the amount or degree ofpolypeptide activity in the cell in the presence and absence of the testagent, wherein an increase or decrease in the amount or degree ofpolypeptide activity in the cell in the presence of the test agentidentifies the test agent as a modulator of the polypeptide's activity.56. The method of claim 55, wherein the cell is an insect cell.
 57. Themethod of claim 56, wherein the cell is a Bombyx mori cell.
 58. Themethod of claim 55, wherein the cell is a plant cell.
 59. The method ofclaim 55, wherein the cell is a mammalian cell.
 60. The method of claim55, wherein the cell is a yeast cell.
 61. The method of claim 55,wherein the degree of polypeptide activity in the cell is determined bymeasuring the amount or degree of apoptosis in the cell.
 62. The methodof claim 55, wherein the degree of polypeptide activity in the cell isdetermined by measuring the amount or degree of caspase proteaseactivity in the cell.
 63. The method of claim 55, wherein the degree ofpolypeptide activity in the cell is determined by measuring the amountor degree of DNA fragmentation in the cell.
 64. The method of claim 55,wherein the degree of polypeptide activity in the cell is determined bymeasuring the amount or degree of cleavage of substrates of caspases inthe cell.
 65. A method of generating an abiotic or bioticinsult-resistant plant comprising the following steps (a) providing anisolated or recombinant polypeptide comprising a sequence having atleast 95% sequence identity to SEQ ID NO:2, wherein the polypeptide iscapable inhibiting apoptosis in a plant cell, and (a) contacting thepolypeptide with the plant in an amount sufficient to inhibit apoptosisin the plant, thereby generating a plant that is biotic insultresistant.
 66. A method for generating an abiotic or bioticinsult-resistant plant comprising the following steps: (a) providing anisolated or recombinant nucleic acid comprising a sequence having atleast 95% sequence identity to SEQ ID NO:1, wherein the nucleic acidencodes polypeptide capable of inhibiting apoptosis in a plant cell, and(b) contacting the nucleic acid with the plant and expressing thenucleic acid to produce an amount of polypeptide sufficient to inhibitapoptosis in the plant.
 67. The method of claim 65 or claim 66, whereinthe biotic insult is induced by a plant pathogen.
 68. The method ofclaim 67, wherein the plant pathogen is a virus, a fungus, a bacteria ora nemotode.
 69. The method of claim 65 or claim 66, wherein the abioticinsult is induced by high moisture, low moisture, salinity, nutrientdeficiency, air pollution, high temperature, low temperature, soiltoxicity, herbicides or insecticides.